Regulation of Microfilament Organization by KaposiSarcoma-associated Herpes Virus-cyclin�CDK6Phosphorylation of Caldesmon*□S

Received for publication, April 11, 2005, and in revised form, August 5, 2005 Published, JBC Papers in Press, August 22, 2005, DOI 10.1074/jbc.M503877200

Maria Emanuela Cuomo‡1, Axel Knebel§¶, Georgina Platt‡, Nick Morrice§, Philip Cohen§, and Sibylle Mittnacht‡2

From the ‡Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research,237 Fulham Road, SW3 6JB London, United Kingdom, §MRC Protein Phosphorylation Unit, University of Dundee, MSI/WTBComplex, Dow Street, DD1 5EH Dundee, United Kingdom, and ¶Kinasource, Laboratory 4.21, MSI/WTB Complex, Dow Street,DD1 5EH Dundee, United Kingdom

Kaposi sarcoma-associated herpes virus (KSHV) encodes aD-likecyclin (K-cyclin) that is thought to contribute to the viral oncoge-nicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4and 6, generating enzymes with a substrate selectivity deviant fromCDK4 and CDK6 activated by D-type cyclins, suggesting differentbiochemical and biological functions. Here we report the identifi-cation of the actin- and calmodulin-binding protein caldesmon(CALD1) as a novel K-cyclin�CDK substrate, which is not phospho-rylated by D�CDK. CALD1 plays a central role in the regulation ofmicrofilament organization, consequently controlling cell shape,adhesion, cytokinesis and motility. K-cyclin�CDK6 specificallyphosphorylates four Ser/Thr sites in the human CALD1 carboxylterminus, abolishing CALD1 binding to its effector protein, actin,and its regulator protein, calmodulin. CALD1 is hyperphosphory-lated in cells following K-cyclin expression and in KSHV-trans-formed lymphoma cells. Moreover, expression of exogenous K-cy-clin results in microfilament loss and changes in cell morphology;both effects are reliant on CDK catalysis and can be reversed by theexpression of a phosphorylation defective CALD1. Together, thesedata strongly suggest that K-cyclin expression modulates the activ-ity of caldesmon and through this the microfilament functions incells. These results establish a novel link between KSHV infectionand the regulation of the actin cytoskeleton.

Nearly all cellular responses are controlled through protein phospho-rylation and the protein kinases catalyzing this process represent thelargest single family of enzymes (1). The choice of substrates determinesthe selectivity of the kinase action and its cellular impact. Therefore, thenature of the substrates of a kinase can provide important clues as to itsphysiological role and function (2).Recently, several oncogenic gamma herpesviruses have been

described to contain within their genome a cyclin-like activator forcyclin-dependent kinases (CDKs)3 (3). TheKaposi sarcomaherpes virus

(KSHV) or human herpes virus 8 (HHV8), a human tumor virus asso-ciated with the development of Kaposi sarcoma and several lymphoidmalignancies in immunocompromised individuals (4–6), encodes acyclin (K-cyclin) that is thought to have descended from cellular D-typecyclins based on co-linearity and sequence identity. Strong evidencefrom transgenic mouse models suggests that K-cyclin contributes sig-nificantly to the oncogenic process elicited by this virus (7, 8).D-type cyclins are recognized for their involvement in human onco-

genesis (9) andK-cyclin shares their ability to activate the closely relatedcellular CDK4 and CDK6 and hence phosphorylate and inactivate theretinoblastoma tumor suppressor protein (Rb) (10).In addition to Rb, K-cyclin�CDK complexes can phosphorylate

proteins that are not substrates for those CDKs when activated bycellular cyclin D. These include the CDK inhibitor p27KIP, which isnormally phosphorylated by cyclin E�CDK2, targeting it for degrada-tion by the proteasome (11, 12). They also include cdc6 and orc1,through which K-cyclin may initiate DNA replication in a manneranalogous to cyclin A (13, 14). A further substrate for K-cyclin�CDKis Bcl2, with consequent loss of its anti-apoptotic function (15). InKSHV negative cells Bcl2 phosphorylation is facilitated by c-JunN-terminal kinase in response to mitotic checkpoint activation (16).These observations suggest that K-cyclin�CDK6 complexes mimicthe activity of a range of other cellular kinases with an impact oncellular functions distinct from cyclin D. The extent to which K-cy-clin-activated CDKs phosphorylate noncanonical substrates is cur-rently unknown.To systematically approach this question, we undertook a kinase sub-

strate tracking and elucidation (KESTREL) screen (17) in which wesearched for proteins phosphorylated by K-cyclin�CDK6. Here, wereport the identification of human caldesmon (hCALD1) as a novelsubstrate for CDK6 kinase in complex with K-cyclin.CALD1 regulates microfilament organization and activities in com-

plex ways (18, 19). In its active form it associates with and cross-linksactin microfilaments. This assists their bundling and stability (20, 21),possibly through interference with actin-severing and -capping activi-ties (22). Other evidence indicates that CALD1 inhibits binding ofArp2/3 to actin, opposing the accelerated filament growth and branch-ing that arise in conjunction with ruffling movement and membraneprotrusion (23). CALD1 can also bind to myosin and in its actin-boundform inhibits the actin-activated ATPase activity of myosin (24).Caldesmon functions are regulated by Ca2�/calmodulin binding and by

* This work was funded by grants from Cancer Research UK (to S. M. and G. P.) and theMedical Research Council (A. K., N. M., and P. C.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) contains supple-mental material.

1 Recipient of an Institute of Cancer Research Ph.D. scholarship.2 To whom correspondence should be sent. Tel.: 44-20-7878-3859; Fax: 44-20-7352-

3299; E-mail: sibylle@icr.ac.uk.3 The abbreviations used are: CDK, cyclin-dependent kinase; Rb, retinoblastoma

tumor suppressor protein; KSHV, Kaposi sarcoma-associated herpes virus; Ct, car-boxyl terminal; IMAC, immobilized metal affinity chromatography; siRNA, smallinterfering RNA; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-

assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spec-trometry; CaM, calmodulin; EGFP, enhanced green fluorescent protein; KESTREL,kinase substrate tracking and elucidation; GST, glutathione S-transferase; ERK,extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase;hCALD1, human caldesmon.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 43, pp. 35844 –35858, October 28, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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phosphorylation, which inhibits actin association and actin-myosinATPase inhibition (25–31). Modulation of CALD1 activity facilitatesthe control of a complex array of cell features including cell shape,cytokinesis, cell adhesion, cell-cell contact, motility, contraction, andthe internal movement of cell organelles (27, 29, 31–35).The results presented here provide novel insight into substrate phos-

phorylation by K-cyclin-associated kinases and further implicate thiscyclin in the regulation of microfilament-associated functions via theectopic phosphorylation and inactivation of caldesmon.

EXPERIMENTAL PROCEDURES

CellCultureandRelatedProcedures—U2OS,NIH3T3,andHeLacellswerecultured inDulbecco’smodifiedEagle’smediumsupplementedwith10%(v/v)heat-inactivated fetal calf serumand4.8mML-glutamineat 37 °Cand5%CO2.KSHV positive PEL cell lines BC-3 (36) and BCP-1 (37) and the EBV immor-talized lymphoblastoid LCL3 were maintained in RPMI 1640 medium with10%fetal calf serum.FrozenHeLacellpelletswerepurchased from4CBiotech.NIH3T3 and U2OS cells were transfected using GenePORTERTM (GeneTherapySystems, Inc.).Totalcell lysateswereproduced in0.25MNaCl,10mM

HEPES-KOH, pH7.0, 5mM �-mercaptoethanol, 10mM �-glycerophosphate,10mMNaF,1mMsodiumorthovanadate,1mMphenylmethylsulfonylfluoride,1mMaprotinin, 2.5�g/ml leupeptin, and 0.5% v/vTritonX-100.

Plasmids and Molecular Cloning—pGEX2T-hRb-(763–928) for theexpression of GST-RbCt (the recombinant carboxyl-terminal fragmentof the human retinoblastoma protein) in bacteria and the constructionof baculovirus vectors for cyclin�CDK expression have been described(38, 39). A full-length cDNA for hCALD1 was produced by reversetranscription-PCR from HeLa cell using RNAzol B (Biogenesis) forRNA preparation and the cDNA cycle kit (Invitrogen) for cDNA syn-thesis. The PCR primers used were 5�-CGCGGATCCATGGAT-GATTTTGAG-3� (forward) and 5� CCGCTCGAGTCAAACCT-TAGTGGC-3� (reverse), covering the full reading frame of humanhCALD1 (Swiss-Prot/GenBankTM accession number Q05682). Tofacilitate directional cloning, primers were designed to introduce arestriction consensus for BamHI adjacent to the start and one for XhoIadjacent to the stop codon (underlined). Following amplification, amajor product was obtained, gel-purified, and inserted into BamHI/XhoI-restricted pcDNA3HisMAX C (Invitrogen) for mammalianexpression or pGEX-6p1 (Amersham Biosciences) for bacterial expres-sion. Clones containing inserts of the correct size were selected andtheir identity validated by DNA sequencing.pcDNA-K-cyclin, pCMV CDK6 and CDK6DN, and expression plas-

mid for the phosphorylation-defective CALD1 (CALD1 7th) have beendescribed (11, 27, 40). pCMV-enhanced green fluorescent protein(EGFP) vector was from Invitrogen.

KESTREL Screen—KESTREL screening was performed essentially asdescribed (17). Briefly, cleared HeLa cell nuclear and cytosolic extracts(equivalent to 15 mg of total protein) were fractionated independentlyusing sequentialMono-Q andMono-S chromatography, and individualfractions were used as substrate for K-cyclin�CDK6 or various controls.Screening for substrates was performed for 5min at 30 °C using 25 �l ofeach fraction and 2 milliunits of cyclin�CDK, or the equivalent amountin protein of the inactive monomeric CDK preparation, in a total vol-ume of 30 �l of KESTREL kinase buffer (4 nM ATP, 5 � 104 cpm of[�-32P]ATP, 40mMTris-HCl, pH 7.5, 2mMMnCl2, 1mMdithiothreitol,aprotinin 20 �g/ml, leupeptin 20 �g/ml). Reactions were terminatedwith 10 �l of SDS-loading buffer (320 mM Tris-HCl pH 6.8, 8% (w/v)SDS, 20 mM EDTA, 32% (v/v) glycerol, 1.14 mM �-mercaptoethanol,

0.02% w/v bromphenol blue) and analyzed using a 7% SDS-polyacryl-amide gel followed by blotting onto a polyvinylidene difluoride mem-brane and exposure to x-ray film.

Purification of p90 K-cyclin�CDK Substrate—The purification of p90is described in the supplement.

Identification of p90 by Mass Spectrometry—Purified p90 was incu-bated for 10 min in the absence or presence of 2 milliunits ofK-cyclin�CDK6 with 10 mMmagnesium and 0.1 mM [�-32P]ATP, dena-tured in SDS, alkylated, loaded on a NuPAGE 4–12% gradient Tris-glycine gel (Novex), and visualized using SYPRO Orange (MolecularProbes). The prominent 32P-labeled SYPRO Orange-stained proteinbandwas excised, digestedwith trypsin, and analyzed bymatrix-assistedlaser desorption time-of-flight mass spectrometry (MALDI-TOF, Per-septive Biosystem Elite STR) as described (17, 41).

Phosphorylation SiteMapping—HeLa cell-derived p90was phospho-rylated for 30 min at 30 °C using 2 milliunits of K-cyclin�CDK6 in 40 �lof KESTREL kinase buffer. 32P-Labeled proteins were digested withtrypsin and the resulting peptides separated by HPLC on C18 resin asdescribed previously. The relative amount of phosphorylation for eachpeptide was estimated from the amount of radioactivity associateddivided by the amount of radioactivity loaded. Phosphopeptides wereanalyzed by MALDI-TOF-TOF mass spectrometry on an Applied Bio-systems 4700 proteomics analyzer, with the peptide sequence and posi-tion of phosphorylation determined byMALDI-MS/MS fragmentationof selected phosphopeptide parent ions. Individual MALDI-MS/MSspectra were searched using the Mascot search engine (MatrixScience)run on a local server. Spectra were also annotated manually. In someinstances phosphopeptides were identified using nanoelectrospraymass spectrometry on aMicromassQ-TOF-2 or anApplied Biosystems4000 Q-Trap mass spectrometer. For independent confirmation, solidphase Edman degradation and one-dimensional phosphoamino acidanalysis was performed as described (17, 42). Residue numbering usedthroughout relates to the sequence of hCALD1 (Swiss-Prot/Gen-BankTM accession number Q05682).

Recombinant Protein Production and Related Procedures—His-hCALD1 was purified from U2OS or NIH3T3 cells transiently trans-fected with pcDNA3HisMAX C-hCALD1 using TALONmetal affinityresin (Clontech). Production and purification of GST-tagged proteinswas as described (38).

In Vitro Protein Phosphorylation—Production of K-cyclin�CDK6,cyclin D1�CDK4, cyclin E1�CDK2, cyclin B1�CDK1, and monomericCDK controls using recombinant baculoviruses was performed asdescribed (38). Specific activities of the different kinase preparations(mol ofATP transfer/mol of substrate)were estimated usingGST-RbCtas a substrate. Unless indicated otherwise, phosphorylation reactionswere conducted in cyclin D-kinase buffer as described previously (38).For routine radioactive reactions, substrates were exposed to kinase for10min at 27 °C in the presence of 10�MATP and 0.1�Ci of [�-32P]ATPin a final volume of 20 �l. Radioactive products were separated on SDS-PAGE and visualized by autoradiography. Signals were quantified byPhosphorImager. Bulk phosphorylated GST-hCALD1 for biochemicalexperiments was produced by incubating 5�g of substrate for 30min at27 °C in a 100-�l reaction containing 1 mM ATP. The amount of kinaseused was optimized to give maximal recognition by the phospho-spe-cific hCALD1 antibodies.For phosphorylation of hCALD1 in the presence of F-actin, F-actin

was preincubated for 30 min on ice with GST-hCALD1 at a 1:1 molarratio in a final volume of 20 �l. 1 �l of kinase and 9 �l of reaction mixcontaining 30�MATP and 0.3�Ci of[�-32P]ATPwere added, and reac-tions were incubated for 10min at 27 °C. Conditions for hCALD1 phos-

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phorylation in the presence of calmodulin (CaM) were identical, exceptthat the ratio of CaM to GST-hCALD1 was 5:1 and the kinase buffercontained 2.5 mM CaCl2.

Antibodies—The antibody reagents used were: mouse monoclonal�-hCALD1 pan antibody C56520 (Transduction Laboratories), mousemonoclonal 9E10 �-myc tag antibody (Hybridoma Unit at CBL, ICR),mouse monoclonal �-actin pan antibody Ab5 (NeoMarker), mousemonoclonal �-human Rb antibody 14001A (BD Pharmingen), rabbitpolyclonal �-CDK6 antibody C-21 (Santa Cruz Biotechnology), mouse�-�-tubulin (Sigma), horseradish peroxidase-conjugated secondaryantibodies (Pierce). Sheep sera with selectivity for hCALD1 phospho-rylated onThr-730 (�-P-hCALD1 730) and Ser-789 (�-P-hCALD1 789)were produced by immunizing sheep with keyhole limpet hemocyanin-coupled phosphopeptides 723CSPTAAG(pT)PNKETA736 (where pT isphosphothreonine) and 782CSVDKVT(pS)PTKV793 (where pS is phos-phoserine), respectively. Sera were affinity-purified by chromatographyon resin-coupled phosphopeptides. For immunoblot analysis, they wereused in the presence of 0.5 �g/ml unphosphorylated peptides.

Calmodulin-Affinity Chromatography—The p90-containing Mono-Qfractionwas loaded onto a 0.5-ml CaM-Sepharose 4B column (AmershamBiosciences) and processed as recommended by the manufacturer. CaMbinding assays using phosphorylatedGST-hCALD1were performed usingCaM-Sepharose 4B, essentially as described (25). Fractions were examinedby SDS-PAGE using SYPROOrange staining or immunoblotting.

Actin Binding Assays—G-actin binding was assessed by incubatingphosphorylated or mock-phosphorylated GST-hCALD1 (1 �g) with 6�M G-actin (Cytoskeleton Inc.) for 30 min at 4 °C in 50 mM HEPES-KOH, pH 7.5, 0.15 MNaCl, 10mMMgCl2, 1mMATP, 10mM �-glycero-phosphate, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothre-itol, 1 mM phenylmethylsulfonyl fluoride, 1% (w/v) aprotinin, 2.5 �g/mlleupeptin, 0.5% v/v Triton X-100). GST-hCALD1 was subsequentlyrecovered on 10 �l of packed glutathione-Sepharose 4B (AmershamBiosciences). Beads were washed three times with 1ml of HEPES bufferand analyzed for associated actin andGST-hCALD1 by SDS-PAGE andimmunoblot.F-actin was prepared using the non-muscle actin-binding protein

spin down assay kit (Cytoskeleton Inc.). F-actin/ligand binding wasmonitored by co-sedimentation following the instructions provided.Supernatant and pellet fractions were collected, resuspended, and ana-lyzed by SDS-PAGE and immunoblot.

SiRNA—BC-3 primary effusion leukemia cells were seeded at a den-sity of 1 � 105 cell/ml into 6-well dishes and transfected with smallinterfering RNA duplexes (siRNA) using HiPerFectTM agent (Qiagen)according to the manufacturer’s instruction. Sequences for targetingthe major latency transcripts L1/L2 (43), which encode K-cyclin, were:5�-AAGUGGUAUUGUUCCUCCUAA-3�, siRNA1; 5�-AATAGCAT-CAATGGTGCCATC-3�, siRNA2. A siRNA pool targeting an irrele-vant, nonessential cell protein (PRKR, SiGenome SMART POOLM-003527-00, Dharmakon) and transfection agent alone were used ascontrols.

Immunofluorescence Microscopy—Cells were fixed 24 h followingtransfection in 4% paraformaldehyde for 5 min at room temperatureand stainedwithTexas Red-X-labeled phalloidin (Molecular Probes) for20 min at room temperature. Fluorescence images were acquired usinga Bio-Rad MRC1024 confocal microscope. The mean phalloidin-asso-ciated fluorescence intensity, cell perimeter, and cell circularity of indi-vidual transfected cells was determined using the ImageJ program(rsb.info.nih.gov/ij/).

RESULTS

KESTREL Screen for Novel Cellular Substrates of K-cyclin�CDK6—The identification of novel target proteins is a powerful means to delin-eate the physiological significance and impact of a given kinase. TheKESTREL methodology, which uses fractionated cellular extract as asubstrate for exogenous purified kinases provides a powerful strategyfor the identification of physiologically relevant protein kinase sub-strates (17, 44–47).We used this methodology to identify novel biological targets for

K-cyclin-activated CDK6. Both nuclear and cytosolic extracts derivedfrom exponentially growing HeLa cells were screened following frac-tionation on either cation or anion chromatography. All resultant frac-tions were assayed in parallel using K-cyclin in complex with humanCDK6 (K-cyclin�CDK6), CDK6 alone (as a negative control), or humancyclin D1 in complex with human CDK4 (cyclin D1�CDK4) for a closelyrelated cellular kinase. All kinase complexes were prepared from bacu-lovirus-infected insect cells. CyclinD activates bothCDK4 andCDK6 tophosphorylate Rb to a similar extent, but cyclin D1�CDK4 complexeshave a higher specific kinase activity when produced in insect cells, andthus this kinase complex was used throughout the KESTREL screen.Reaction conditions were chosen such that a common substrate forK-cyclin�CDK6 and cyclin D1�CDK4, Rb Ct (amino acids 763–928), wasphosphorylated equally by both of the enzymes (Fig. 1A). No incorpo-ration of phosphate into Rb Ct was observed with CDK6 alone, indicat-ing that a kinase activity resembling K-cyclin�CDK6 is not generated ininsect cells in the absence of K-cyclin expression. To distinguish phos-phorylation products that may derive from contaminants in the kinasepreparation or by autophosphorylation of the kinase itself, a set of reac-tions was run in the absence of exogenous substrates (see for exampleFig. 1B, lanes 1–3). Lastly, all fractions were assayed without exogenouskinase preparation in order to identify signals arising from phosphoryl-ation by HeLa cell-derived kinases (see for example Fig. 1B, lane 7). Wenote that omission of exogenous kinase often yielded phosphorylationactivity that was not seen when kinase preparation had been added tothe fraction, suggesting that one or more components of the insectcell-derived kinase preparations may inhibit many of the endogenouskinase activities present in the column fractions (compare for examplelanes 4–6 with lane 7 in Fig. 1B).In total, 80 different HeLa-derived fractions were screened yielding

evidence for a minimum of four distinct substrates that were phospho-rylated specifically in K-cyclin�CDK6- but not cyclinD1�CDK4-contain-ing reactions (not shown). This confirms the known ability of K-cyclinto modulate CDK substrate selection toward a broader range of sub-strates when compared with cellular cyclin D. In addition, two sub-strates specifically phosphorylated in vitro by cyclin D1�CDK4 but notK-cyclin�CDK6 were discovered. This could suggest that diversion ofK-cyclin from cyclin D generates a kinase activity that does not simplyfacilitate phosphorylation of more but instead a different set of sub-strates (not shown).Fig. 1B shows the phosphorylation pattern of Mono-Q fraction 8

from nuclear extract, revealing a putative protein substrate with anapparent molecular mass of 90 kDa (p90) that is strongly phosphoryl-ated in reactions containing K-cyclin�CDK6 (lane 5). Phosphorylationof p90 is not apparent in samples containing CDK6 alone (lane 4) orcyclin D�CDK4 (lane 6) or in the sample containing K-cyclin�CDK6 inthe absence of added substrate, inferring that p90 is derived from HeLacells and phosphorylated selectively by K-cyclin�CDK6.

Identification of p90—To determine the identity of p90, we purifiedthis putative substrate fromHeLa nuclear extracts employing sequentialchromatography on Mono-Q, heparin-Sepharose, and Mono-S (see

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supplemental Fig. S1) and phosphorylation by K-cyclin�CDK6 to trackits location on each column. p90 substrate positive fractions fromMono-S were pooled and loaded on a gradient gel followed by SYPROOrange staining. The major product in this preparation was a 90-kDaprotein (Fig. 1C), which co-migrated with radiolabeled K-cyclin�CDK6-phosphorylated p90 (not shown). Tryptic fingerprinting of this proteinyielded peptidemassesmatchingwith hCALD1with sequence coverageof 25% (summarized in supplemental Fig. S2). This suggested that the90-kDa K-cyclin�CDK6 substrate is human caldesmon.To substantiate that the p90 substrate and caldesmon were the same

protein, we tested whether p90 from the Mono-Q column (see Fig. 1B)was retained in a Ca2�-dependentmanner on a CaM affinity column, aswould be expected for CALD1, which is known to bind Ca2�/CaM. p90effectively bound to the resin in the presence of Ca2�, being undetect-able in either the flow-through (FT) or wash fractions (W1, W2) butreadily detectable in the eluate when using EGTA-containing buffer(E1, E2, E3) (Fig. 1D). This finding strongly supported the assignmentmade on the basis of the tryptic mass fingerprinting.

Identification of Phosphorylation Sites by Mass Spectrometry—Toconfirm that K-cyclin�CDK6 phosphorylates p90-hCALD1 and to iden-tify the sites of phosphorylation, we processed the HeLa-derived p90after incubation with K-cyclin�CDK6 for identification of phosphoryl-ated peptides. Separation on reverse phase chromatography of the tryp-sin digest of purified p90 substrate showed twomajor radioactive peaks

(Fig. 2A, P1 and P2).Mass spectrometry analysis revealed that each peakcontained a single phosphopeptide with masses consistent withhCALD1-derived tryptic peptides comprising residues 782–792 (P1)and residues 719–739 (P2). One-dimensional phosphoamino acid anal-ysis demonstrated modification on serine for P1 and threonine for P2,and solid phase Edman degradation confirmed that the sites of phos-phorylation were on residue 8 for P1 and residue 12 for P2, correspond-ing to Ser-789 and Thr-730, respectively (Fig. 2, C and D). The moreminor release of 32P at residue 8 for peptide P2 (Fig. 2D) could representthe phosphorylation of threonine 726, but there was no mass spectralevidence to suggest that the diphosphopeptide phosphorylated at boththreonine 726 and threonine 730 was present. Both Thr-730 and Ser-789 comply with the (S/T)P consensus known to be required for thephosphorylation by CDK4/6 kinases, thus providing strong evidencethat hCALD1 is a direct substrate for K-cyclin�CDK6. The radioactivityassociated with the respective peaks accounted for 40% (P1) and 46%(P2) of the total incorporated radioactivity, suggesting that Thr-730 andSer-789 are the major K-cyclin�CDK6 phosphorylated sites in hCALD1purified from exponentially growing HeLa cells.

K-cyclin Phosphorylation of CALD1 in Vitro—To provide independ-ent evidence that hCALD1 is a direct substrate for K-cyclin�CDK6, wegenerated two recombinant forms of human caldesmon. A constructwas engineered for the expression in Escherichia coli of full-lengthhCALD1 as a GST-tagged protein, allowing purification of the product

FIGURE 1. Identification of caldesmon as a novelK-cyclin�CDK6 substrate by KESTREL screening.A, K-cyclin�CDK6 phosphorylates Rb in the pres-ence of Mn2�ATP. The CDK complexes indicatedwere used to phosphorylate recombinant GST-RbCt (amino acids 763–928) as substrate. Reactionproducts were separated by SDS-PAGE on 7% gels,transferred to a polyvinylidene difluoride mem-brane, and autoradiographed. B, detection of aK-cyclin�CDK6-specific substrate in HeLa cells.HeLa cell nuclear extract was chromatographedon Mono-Q, and aliquots of each fraction collectedwere phosphorylated in the absence (lane 7) orpresence (lanes 4 – 6) of the indicated cyclin�CDKcomplexes, matched for activity toward GST-Rb Ct.Control reactions without HeLa extract were runfor each cyclin�CDK complex (lanes 1–3). Reactionproducts were processed as described in A. Thefigure shows the results for fraction 8 from theMono-Q column, where the 90-kDa substrate wasdetected. C, purification of the p90 substrate. Thep90 protein was purified as described under”Experimental Procedures,“ subjected to SDS-PAGE on a 4 –12% gradient gel, and stained withSYPRO Orange. D, the p90 protein (caldesmon)binds to calmodulin-Sepharose. p90 (from B) wassubjected to chromatography on calmodulin-Sepharose. Aliquots of the elution buffer (�, neg-ative control), flow-through (FT), washes (W1, W2),and successive EGTA eluates (E1, E2, and E3) werethen phosphorylated in the presence ofK-cyclin�CDK6.

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by affinity chromatography on glutathione-Sepharose. Furthermore, wegenerated a construct for mammalian cell expression of a hexahistidine(His)-tagged hCALD1, permitting purification of the product by immo-bilized metal affinity chromatography (IMAC). Using purified versionsof these proteins as substrates, we probed for phosphorylation byK-cyclin�CDK6 together with other cyclin�CDKs (Fig. 3,A and B). Reac-tions containing GST-Rb Ct, a common substrate for these kinases,were run in parallel as a reference for the relative level of activity asso-ciated with each kinase (Fig. 3A, lower panels). Comparable amounts ofGST-hCALD1 and His-hCALD1 were present in each sample as deter-mined by Western blot (Fig. 3A, top right panel; Fig. 3B, bottom panel).Both forms of recombinant hCALD1 were avidly phosphorylated byK-cyclin�CDK6, corroborating the proposition that human caldesmonconstitutes a genuine substrate for this kinase in vitro. Consistent with

results from the KESTREL screen, recombinant hCALD1was not phos-phorylated by cyclinD1�CDK4, although this kinase effectively phos-phorylated the GST-Rb Ct reference substrate. However, hCALD1 wasphosphorylated by cyclin A2�CDK2, cyclin A2�CDK1, cyclin B1�CDK1,and to a lesser extent by cyclin E1�CDK2. It has previously been reportedthat purified mitotic HeLa cell CDK1 can phosphorylate CALD1 (34,48–50) and that this may be critical for the induction of microfilamentdisassembly during mitosis (48, 51). However, phosphorylation ofCALD1 by cyclin A and E-associated kinases, which gain activity duringG1 and S phase, has not been reported previously.

We further assessed whether phosphorylation of recombinanthCALD1 arises at Thr-730 and Ser-798 by immunoblotting using phos-pho-specific antisera to these sites. Neither �-P-hCALD1 730 nor �-P-hCALD1 789 serum recognized GST-hCALD1 exposed to inactive,

FIGURE 2. Identification of residues phosphorylated by K-cyclin�CDK6 in HeLa cell caldesmon. A, separation of 32P-labeled tryptic peptides of caldesmon by reverse phasechromatography. Purified hCALD1 was phosphorylated by K-cyclin�CDK6 in the presence of radioactive ATP. Following SDS-PAGE the 32P-labeled band, corresponding to full-lengthhCALD1, was excised and digested with trypsin. Peptides were then separated on a C18 resin equilibrated in 0.1% (v/v) trifluoroacetic acid and developed with an acetonitrile gradient(broken line). The elution profile was monitored for 32P radioactivity (solid line). B, phosphoamino acid analysis of peptides P1 and P2 from A. Each peptide was hydrolyzed, andphosphoamino acids were separated by thin layer chromatography and detected by autoradiography. Phosphoamino acids standards are indicated. C and D, identification of thesites of phosphorylation in peptides P1 (C) and P2 (D). Analysis by mass spectrometry indicated that P1 and P2 correspond to residues 782–792 and 719 –739, respectively. Eachpeptide was subjected to solid phase sequencing to identify the cycle of Edman degradation at which 32P radioactivity was released from the peptide.

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monomeric CDK6, but both antibodies gave signals with hCALD1phosphorylated in the presence of K-cyclin�CDK6, cyclin A�CDK2,cyclin E�CDK2, and cyclin B�CDK1, but not cyclin D1�CDK4, althoughcomparable amounts of hCALD1 were present in each sample (Fig. 3C,top panel). Recognition of phosphorylated caldesmon by these antibod-ies is dependent on the presence of phosphate-accepting residues attheir cognate recognition site (supplemental Fig. S3). This demon-strated the site-selective and phosphorylation-dependent recognitionof hCALD1 by these reagents and established that K-cyclin�CDK6, aswell as cellular cyclin A, E, and B-activated CDKs, can phosphorylaterecombinant hCALD1 on Thr-730 and Ser-789 in vitro.

K-cyclin Phosphorylation of hCALD1 in Vivo—In vitroCALD1 can bephosphorylated by several serine/threonine protein kinases, includingcyclin B2�CDK1 (34), Ca2�/calmodulin kinase II (52), casein kinase II(53–55), p21-activated kinase (56), protein kinase C (57, 58), and extra-cellular signal-regulated kinases (ERK) (59, 60). However, evidence forCALD1 phosphorylation in cells has been provided only for themitosis-activated CDK1 in non-muscle cells (25, 48) and ERK in smoothmuscle(59–61).

To address whether hCALD1 is capable of being phosphorylated byK-cyclin�CDK6 in cells, we transiently co-expressed His-hCALD1,K-cyclin, and CDK6 in osteosarcoma-derived U2OS, which do not con-tain the KSHV sequence to express K-cyclin, and investigated the phos-phorylation state of hCALD1. Analysis of cell lysates showed that His-hCALD1 was expressed adequately and to a similar level, regardless ofthe presence of K-cyclin�CDK6, and further, that K-cyclin and CDK6were expressed correctly (Fig. 4A). After IMAC purification of lysates,analysis of the His-hCALD1 using phosphorylation-selective hCALD1antibodies demonstrated that Thr-730 is not detectably phosphorylatedin cells in the absence of K-cyclin�CDK6 but that a strong signal isobserved when cells express K-cyclin�CDK6 (Fig. 4B,middle panel). Byperforming immunoblots of serial dilutions of each sample (2-fold lowereach time), we showed that phosphorylation of Thr-730 increases bymore than 8-fold, thus indicating a major effect of K-cyclin�CDK6 onthemodification of this site (Fig. 4B, top andmiddle panels). In contrast,Ser-789 was detectably phosphorylated in cells that did not expressK-cyclin�CDK6, in line with reports that Ser-789 is prominently modi-fied in cells (60, 61). Despite this finding, some increase is observed in

FIGURE 3. Recombinant caldesmon is a substrate for K-cyclin�CDK6. A, phosphorylation of bacterially produced human caldesmon by cyclin�CDK complexes. GST-hCALD1 andGST-Rb Ct were phosphorylated in parallel reactions with [�-32P]ATP and the indicated kinase complexes, subjected to SDS-PAGE, and autoradiographed (left panels). Aliquots fromthe kinase reactions were analyzed by Western blot for the presence of substrate (right panels). B, phosphorylation of caldesmon produced in mammalian cells by cyclin�CDKcomplexes. Hexahistidine-tagged hCALD1 produced by transient transfection in NIH3T3 cells from the His-pcDNA-hCALD1 vector was purified by IMAC and phosphorylated asdescribed in A (top panel). Lysate from His-pcDNA vector-transfected cells served as a negative control. The presence of His-CALD1 protein in each sample was tested by Western blot(bottom panel). C, phosphorylation of recombinant hCALD1 on Thr-730 and Ser-789. 0.5 �g of recombinant GST-hCALD1 was phosphorylated by the cyclin�CDK complexes indicated.Reactions were separated by SDS-PAGE, blotted onto a membrane, and immunostained with antibody �-hCALD1 against pan-hCALD1 and with phospho-specific antibodies�-P-hCALD1 730 and �-P-hCALD1 789, respectively.

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cells containing K-cyclin�CDK6 (Fig. 4B, bottom panel). Taken together,these results support the notion that K-cyclin�CDK6 complexes canmodulate phosphorylation of hCALD1 in cells and do it both quantita-tively, by increasing the level of base-line phosphorylation, and qualita-tively, by introducing a modification that is not readily found in theabsence of this kinase complex.To extend our analysis, we compared the phosphorylation state of endoge-

nous hCALD1 in KSHV negative (LCL3) and KSHV positive (BC-3 andBCP-1) pre-B cell-derived lymphoblastoid cell lines. Both BC-3 and BCP-1

express K-cyclinmRNAand protein, whereas these are undetectable in LCL3(62). Comparative analysis of lysates derived from these cell lines using �-P-hCALD1 730 antibody revealed that this site was barely phosphorylated inLCL3 but was considerably modified in the KSHV positive BC-3 and BCP-1cells (Fig. 4C,middle panel).Modification of Ser-789 did not rise asmarkedly,consistentwith thenotion that this sitemay already bemodified constitutivelyatahighlevel inKSHVnegativecells,andonlyaminorincreasewasapparentinthe KSHV positive lines (Fig. 4C, bottom panel). Thus, in cells with naturalK-cyclinexpression,wedetectedachangeofhCALD1phosphorylation,which

FIGURE 4. Phosphorylation of caldesmon by K-cyclin�CDK6 in vivo. A, co-expression of recombinant caldesmon and K-cyclin�CDK6 in mammalian cells. U2OS cells were transfectedwith His-pcDNA-hCALD1 or His-pcDNA together with plasmids encoding K-cyclin and CDK6. After 48 h, cells were lysed, and expression of relevant proteins was assayed byimmunoblotting using the indicated antibodies. B, recombinant caldesmon is phosphorylated by K-cyclin�CDK6. The lysates from A were purified by IMAC, and serial dilutions (2-foldin successive lanes) were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted using �-hCALD1, �-P-hCALD1 730, or �-P-hCALD1 789 antibodies. C, endogenouscaldesmon is hyperphosphorylated in KSHV positive lymphoma lines. Equivalent amounts of lysate from two KSHV positive cell lines (BC3 and BCP1) and a KSHV negative lympho-blastoid line (LCL3) were probed using �-hCALD1, �-P-hCALD1 730, or �-P-hCALD1 789 antibodies as indicated. Lysates from 0.5 � 105 and 1 � 105 cells, respectively, were loadedin successive lanes to allow easy comparison of the relative signal levels. D, down-regulation of K-cyclin expression reduces CALD1 phosphorylation. BC-3 cells were transfected withsiRNA targeting independent regions of the K-cyclin-encoding transcripts (siRNA 1 and 2), an irrelevant siRNA (siRNA C), or mock-treated using transfection agent alone (mock). Lysateswere prepared 48 h after transfection and probed with antibodies as indicated. Numbers below the top and middle panels denote the average intensity of the K-cyclin and P-Thr-730hCALD1-associated autoradiography signal as determined by the ImageJ program. Consistent results were obtained in two independent experiments.

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in degree and appearance is greatly reminiscent of that seen with exogenousexpression of K-cyclin�CDK6. To further confirm the link between theenhancedCALD1 phosphorylation andK-cyclin, wemade use of RNA inter-ference to diminish K-cyclin protein expression in BC-3 PEL cells (Fig. 4D).Both siRNA1 and siRNA2, designed to target K-cyclin-encoding transcripts,down-regulatedK-cyclin protein by about 80 and 50%, respectively, and treat-mentwithbothreducedphosphorylationofCALD1Thr-730whencomparedwithmocktreatedcells. Incontrast,anirrelevantcontrolsiRNA(siRNAC)hadno effect. None of the siRNA oligonucleotides used affected the levels ofCALD1 protein itself. Phosphorylation of Ser-789 was also not detectablyaffected, in accordancewith thepreviousobservation thatmodificationof Ser-789 may be largely K-cyclin-independent. Equal loading of protein was dem-onstrated with antibodies against �-tubulin (Fig. 4D, bottom panel). Thus,down-regulation of K-cyclin in KSHV-infected cells results in a specific alter-ation of CALD1 phosphorylation status, providing additional confidence thatK-cyclin candrive caldesmonmodification in vivo. Together, the above exper-imentsprovidestrongevidencethathCALD1isabonafide invivosubstrate forK-cyclin-activated kinase.

Saturation Mapping of Phospho-sites on Recombinant HumanCaldesmon—Our previous results (Fig. 2) showed that K-cyclin�CDK6phosphorylates HeLa-derived p90 substrate on two major sites. How-ever, six sites complying with the canonical consensus (S/T)P are pres-ent in hCALD1, five of which are conserved in homologues from othermammalian species (see Fig. 5E) (48). Furthermore, previous work onmitotic CDK1-phosphorylated CALD1 using either Edman sequencing(49) or mutagenesis (63) provided evidence for in vitro phosphorylationof sites in addition to Thr-730 and Ser-789.Therefore, using recombinant GST-hCALD1 as a substrate, we

reevaluated the extent of possible phosphorylation by K-cyclin�CDK6.Phosphorylation of GST-hCALD1 resulted in near maximal incorpora-tion of phosphate after 30 min (Fig. 5A). C18 chromatography of trypticdigests derived from this material resolved into sevenmajor 32P-labeledpeaks (P1-P7) (Fig. 5B), together accounting for more than 90% of thetotal radioactivity incorporated and, hence, likely to account for the fullcomplement of phosphorylated residues. Mass spectrometry analysisunambiguously located the peptides corresponding to six of the peakswithin the hCALD1 sequence, together with the identification of thephosphorylated residues (for summary see Fig. 5C). P1 and P2 peptideswere identical to P1 andP2 as observed and characterized in our analysisusing cell-derived hCALD1 (see Fig. 2), thus confirming phosphoryla-tion of the recombinant hCALD1 on Thr-730 and Ser-789. The identityof the peptide producing peak P3 could not be determined by eithermass spectrometry or Edman sequencing, raising the possibility that itrepresents a very small peptide species that evades detection by thesemethods. P4 and P5were found to contain peptides related to P1 and P2,phosphorylated on Thr-730 and Ser-789, respectively. P6 and P7 wererelated peptides with masses that predicted phosphorylation at bothThr-753 and Ser-759. Both of these peptides contain trypsin-missedcleavages between Lys-752 andThr-753 andLys-758 and Ser-759 due tothe presence of a phosphorylated residue next to the site of trypticcleavage. Both peptides also contain the oxidized form of tryptophan(kynurenin), and interestingly, the P6 peptide was generated by anunusual tryptic cleavage across the Lys-762–Pro-763 bond, which isnormally trypsin-resistant. The sites of phosphorylation were con-firmed by both MALDI-MS/MS (not shown) as well as solid phaseEdman degradation, which revealed releases of radioactivity followingcycle 8 and cycle 14 for each peptide (Fig. 5D).Together, our analyses reveal that, as for the cell-derived hCALD1

substrates, Thr-730 and Ser-789 are major sites of phosphorylation in

recombinant hCALD1. In addition, Thr-753 and Ser-759 are modifiedin recombinantmaterial (see Fig. 5E for summary). It is notable that 45%of the total radioactivity was associated with peaks P6 and P7, whichcover Thr-753 and Ser-759, indicative that phosphorylation on thesesites occurs at a stoichiometry similar to that of Thr-730 and Ser-789.Why Thr-753 and Ser-759 are not apparently phosphorylated in cell-derived hCALD1 is not clear, but it may be because of prior phospho-rylation of these sites by cell-derived kinases, dephosphorylation byphosphatases contaminating the cell-derived hCALD1 preparation,and/or incomplete phosphorylation of the substrate under the condi-tions used. We noted that all sites identified in the recombinanthCALD1 conformed to the known consensus for phosphorylation byCDK.The array of sites modified by K-cyclin�CDK6 in recombinant

hCALD1 closely resembles, but is not identical to, those reported for rator chicken caldesmon phosphorylated by mitotic CDK1 (63). In thesestudies CDK1 was found to modify Ser-724, which apparently is notphosphorylated by K-cyclin�CDK6. Although Ser-724 is containedwithin peptides yielding peak P2 from both cell-derived and recombi-nant hCALD1 and P5 from recombinant hCALD1, it is always detectedin an unphosphorylated state, suggesting that itmay not be used as a sitefor modification by K-cyclin�CDK6. In chicken CALD1, mitotic CDK1also targets a site corresponding to Thr-638 in the human sequence. It ispossible that P3, which evaded identification in our analysis, contains apeptide with modification on this residue. The small size of this pre-dicted tryptic peptide, CFTPK, is in line with such an assumption.

Effects of K-cyclin�CDK6Phosphorylation on hCALD1Function—Theresults presented above indicate that K-cyclin�CDK6 is capable of tar-geting the majority of sites known to become modified upon CALD1phosphorylation bymitotic CDK1 in vitro. Importantly, these sites clus-ter within the regions known to facilitate binding of CALD1 to actin (seeFig. 5E) (64–67), and phosphorylation of CALD1 by mitotic CDK1 (25,63) and ERK1 (28, 30) has previously been shown to affect thisinteraction.We therefore investigated the impact of K-cyclin�CDK6 phosphoryl-

ation on binding of GST-hCALD1 to actin filaments using the knownability of CALD1 to associate and co-sediment with filamentous (F-)actin. Consistent with this, the majority of GST-hCALD1 was drawninto the pellet fractionwhen centrifuged in the presence of F-actinwhileremaining in the supernatant when on its own (Fig. 6A). Co-sedimen-tation with F-actin was unaffected when CDK6 alone was used duringthe phosphorylation reaction. In contrast, GST-hCALD1 phosphoryl-ated in the presence of K-cyclin�CDK6 or cyclin B�CDK1 remained inthe supernatant, thus providing evidence that phosphorylation byK-cyclin�CDK6 disables the association of hCALD1with actin filamentsas it does phosphorylation by cyclin B�CDK1.In addition to its ability to interact with F-actin, which results in actin

filament bundling and actin-myosin cross-linking, CALD1 can bind tomonomeric (G-) actin (68). The latter is thought to facilitate nucleation,which is instrumental when rebuilding the actin cytoskeleton followingmitosis and during cell movement. Tomonitor the interaction betweenCALD1 and G-actin, we performed pull-down assays using GST-hCALD1 bound to a glutathione-Sepharose matrix (Fig. 6B). TheSepharose bound GST-hCALD1 permitted recovery of G-actin fromsolution (lanes 5 and 6), whereas GST-Rb Ct used in the same condi-tions did not (lanes 7 and 8). Importantly, binding of G-actin to GST-hCALD1 was abolished after phosphorylation with K-cyclin�CDK6 orcyclin B�CDK1, but not monomeric CDK6 (Fig. 6C, top panel). Notethat comparable amounts of hCALD1 protein were recovered on thebeads, as shown by immunoblotting using a �-hCALD1 pan antibody

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FIGURE 5. Identification of residues phosphorylated by K-cyclin�CDK6 on recombinant caldesmon. A, phosphorylation of bacterially produced hCALD1 by K-cyclin�CDK6 in vitro.0.5 �g of purified GST-hCALD1 produced in E. coli was incubated with K-cyclin�CDK6 in the presence of [�-32P]ATP. After separation on SDS-PAGE, the full-length GST-hCALD1 wasexcised from the gel and the specific radioactivity determined by Cerenkov counting. B, separation of 32P-labeled tryptic peptides from GST-hCALD1 using reverse phase HPLC.Purified GST-hCALD1 was phosphorylated by K-cyclin�CDK6 in the presence of [�-32P]ATP and analyzed as described in the legend for Fig. 2A. C, analysis of phosphopeptides derived

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(Fig. 6C, middle panel). Taken together, the above data demonstratethat phosphorylation by K-cyclin�CDK6 abolishes the interaction ofhCALD1 with both monomeric and filamentous actin, inferring thatthis kinase affects all known mechanisms by which caldesmon modu-lates the function of microfilaments.

Effects of K-cyclin�CDK6 Phosphorylation on CALD1 Binding toCalmodulin—CALD1 also interacts with Ca2�/CaM, and this interac-tion is thought to constitute a regulatory event preventing de novo bind-ing of free CALD1 to monomeric and filamentous actin. Yet other evi-dence exists that association of Ca2�/CaM with filament-bound

from caldesmon by mass spectrometry. Shown are the masses [M�H]� observed by MALDI-TOF-TOF mass spectrometry compared with the theoretical values calculated from theamino acid sequences #, peptide P4 was analyzed by nanoelectrospray mass spectrometry, and the mass values represent that of the [M�2H]2� ion. s, phosphoserine; t, phospho-threonine; w, kynurein. D, identification of the phosphorylation sites in peptides P6 and P7. Peptides P6 and P7 from B were subjected to solid phase sequencing as described for Fig.2, C and D. E, functional domains of hCALD1. The myosin-binding domain is shown as cross-hatched bars, the regions that bind actin (amino acids 630 –734 and 749 –790) andcalmodulin (amino acids 715–722 and 744 –752) are shaded gray and black, respectively. The (S/T)P consensus sequences are indicated. Positions phosphorylated by K-cyclin�CDK6are marked with an asterisk.

FIGURE 6. Reduced binding to actin of caldesmon phosphorylated by K-cyclin�CDK6. A, binding of caldesmon to actin filaments. Unphosphorylated GST-hCALD1 or GST-hCALD1phosphorylated with the indicated cyclin�CDK complexes was incubated for 30 min at room temperature in the absence (�) or presence (�) of F-actin. Following centrifugation for1 h at 150,000 � g, the resulting supernatant (S) and pellet (P) fractions were resuspended in an equivalent volume of sample buffer, subjected to SDS-PAGE, and analyzed byimmunoblotting with the indicated antibodies. B, binding of caldesmon to G-actin. GST-hCALD1 was incubated for 30 min at 4 °C with G-actin and bound proteins recovered onglutathione-Sepharose 4B. Material bound on the beads was separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. The GST-Rb Ct protein was usedas negative control. C, binding of phosphorylated caldesmon to G-actin. GST-hCALD1 was mock-phosphorylated or phosphorylated as described in A, incubated with G-actin, andprocessed as described in B.

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CALD1may result in stabilization of existing filaments (31). Ca2�/CaMbinds to a discontinuous sequence that overlaps with the CALD1 actinbinding sites (69) and is positioned adjacent to the residues modified byK-cyclin�CDK6 (see Fig. 5E).We thus investigated whether phosphorylation by K-cyclin�CDK6

may affect the ability of hCALD1 to associate with calmodulin bymeas-uring the retention of phosphorylated and unphosphorylated GST-hCALD1 on CaM-Sepharose in the presence of Ca2� (Fig. 7). GST-hCALD1was detected using either SYPROOrange staining or pan- andphospho-site -selective hCALD1 antibodies. In its unphosphorylatedform or when treated with monomeric CDK6, GST-hCALD1 boundfirmly to the resin and was released only with EGTA-containing elutionbuffer. In contrast, when phosphorylated by K-cyclin�CDK6 or cyclinB�CDK1, GST-hCALD1 eluted during the initial wash. Thus, phospho-rylation by K-cyclin�CDK6 or cyclin B�CDK1 substantially reduces theaffinity of hCALD1 for calmodulin. In summary, phosphorylation ofhCALD1 by K-cyclin�CDK6 broadly affects the biochemical propertiesof CALD1, disabling its interaction both with its effector (actin) andwith its regulator (Ca2�/CaM).

Effects of Actin and Calmodulin on K-cyclin�CDK6-mediated Phos-phorylation of hCALD1—Previous work showed that association ofCALD1 with F-actin or Ca2�/CaM blocks the phosphorylation ofCALD1 bymitotic CDK1 (50), suggesting that this kinasemay primarilyact on free CALD1 and does not affect filament- or Ca2�/CaM-boundforms.Because the pattern of hCALD1 phosphorylation by K-cyclin is sim-

ilar to that by cyclin B�CDK1, we investigated whether F-actin or cal-modulin binding affects phosphorylation by K-cyclin�CDK6. As shownin Fig. 8A, preincubation with F-actin reduced the phosphorylation ofhCALD1 by K-cyclin�CDK6 by more than 60%, affecting this kinasemore substantially than did cyclin B�CDK1. Similarly, pre-binding toCa2�/CaM also partially suppressed K-cyclin�CDK6 phosphorylation ofhCALD1 (Fig. 8B), although the impact was less pronounced than thatof actin and similar in degree for K-cyclin�CDK6 and cyclin B�CDK1.These results indicate that phosphorylation of hCALD1 byK-cyclin�CDK6 and cyclin B�CDK1 follows similar restrictions, indi-rectly suggesting that similar conformational requirements may existfor recognition or access to the phosphate-accepting amino acids forboth cyclin�CDK complexes.

K-cyclin Expression Affects Microfilament Integrity and Cell Shape—Previous work has implicated CALD1 in the regulation of microfila-

ments integrity in vivo (27).We therefore probed for the effects of K-cy-clin expression on actin cytoskeleton morphology using human osteo-sarcoma-derived U2OS cells. These cells, which display extensive stressfibers, were transiently transfected with a plasmid encoding K-cyclin orempty vector, together with a plasmid encoding EGFP to mark trans-fected cells. After 24 h, phalloidin staining of F-actin revealed remarka-ble alterations in microfilament appearance in K-cyclin-transfected butnot in empty vector-transfected cells (Fig. 9A). These changes includedthe near absence of cortical actin and stress fibers in K-cyclin-express-ing cells, which instead presented with short fiber fragments. Quantita-tive analysis (Fig. 9B) involving optical scoring of 100 cells per conditionin three independent experiments revealed thatmore than 60% of K-cy-clin-transfected cells lacked the normal, linear appearance of actin bun-dles but instead showed fragmented filaments. In contrast, nearly 95%ofcells transfected with empty vector contained linear actin bundles, andcells with fragmented or undetectable filaments were rare in these sam-ples. The effects on microfilament appearance elicited by K-cyclinexpression were almost fully overcome when a kinase-defective, domi-nant negative form of CDK6 (CDK6DN) or a mutant form of CALD1with alanine substitutions in its proline-directed phosphorylation sites(CALD1 7th) was co-expressed but not when catalytically active CDK6or wild-type caldesmon was used instead (Fig. 9B). Representative pho-tomicrographs of cells transfected with the various plasmid combina-tions are shown in supplemental Fig. S4. Results in accord with thesewere obtained when phalloidin fluorescence in individual cells wasquantified using the mean intensity algorithm provided by the ImageJsoftware (Fig. 9C). Cells from K-cyclin-transfected cultures displayedsubstantially lower mean fluorescence intensity, consistent with theabsence of fibers or substantially decreased fiber density. The decreasein mean intensity was abolished by co-expression of CDK6DN or phos-phorylation-defective CALD1, which partially, following expression ofwild-type CALD1, provided direct evidence that K-cyclin can affectmicrofilament organization by amechanism involvingCDKactivity andcaldesmon phosphorylation. Consistent with previous results (27), bothwild-type and phosphorylation defective CALD1 when expressed inisolation resulted in an increase in mean fluorescence intensity, in linewith CALD1 stabilization of actin filament formation and density.Together, the above results strongly support the notion that K-cyclinthrough CDK-dependent phosphorylation of caldesmon affects micro-filament organization and structure.

FIGURE 7. Reduced binding to calmodulin of caldesmon phosphorylated by K-cyclin�CDK6. 3 �g of either unphosphorylated GST-hCALD1 or GST-hCALD1 phosphorylated bythe indicated cyclin�CDK complexes in vitro were chromatographed separately onto a calmodulin resin. Fractions collected from the washing and elution steps (indicated as W1 andW2 and E1, E2, E3, and E4, respectively) were subjected to SDS-PAGE and stained for protein with SYPRO Orange. Further aliquots were also immunoblotted with �-hCALD1 and�-P-hCALD1 730 antibodies, as indicated. Seph, Sepharose.

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The observed modification in the actin cytoskeleton was accompa-nied by changes in cell shape. U2OS cells normally feature a symmetri-cal polygonal shape and grow in tight, epithelium-like clusters. Thesefeatures were unaltered in cells transfected with empty control vector.In contrast, cells expressing K-cyclin displayed highly irregular shapeswith an increased number of membrane protrusions, an elongatedappearance, and an overall loss of cell-cell contact (see Fig. 9A andsupplement Fig. 4). These observations were confirmed by measure-ments based on the assessment of cell circularity and cell perimeter inindividual transfectants. Such measurements indicate a consistent lossof circularity (Fig. 9D) in the K-cyclin-expressing cells but an increase incell perimeter (Fig. 9E). As in the previous analysis, co-expression ofeither CDK6DN or phosphorylation-defective CALD1 7th, but not thewild-type version of these proteins, abolished the K-cyclin associatedeffects, providing strong evidence that, likemodulation of themicrofila-ment structure, these shape responses also relate to the CDK-mediatedphosphorylation of caldesmon.Taken together, the above results support the notion that K-cyclin

affects the integrity of actin stress fibers and, through this, cellular mor-phology by targeting caldesmon for CDK6 phosphorylation.

DISCUSSION

We provide evidence here that the actin- and calmodulin-bindingprotein caldesmon is a substrate for CDK6 when activated by K-cyclin,the D-like cyclin encoded by Kaposi sarcoma herpes virus. We demon-

strate that K-cyclin�CDK6 complexes phosphorylate and modify theproperties of recombinant hCALD1 in vitro and that K-cyclin expres-sion promotes and qualitativelymodulates hCALD1 phosphorylation incells. Furthermore, expression of K-cyclin in culturedU2OS cells affectsactin cytoskeleton integrity and cellular shape; CDK catalysis and phos-phorylation of caldesmon are essential for both these responses.Together, these results provide strong evidence that K-cyclin has thecapability to affect caldesmon activity and through this modulatemicrofilament functioning and associated events in cells.Evidence for the phosphorylation of hCALD1 by K-cyclin�CDK com-

plexes came from a KESTREL screen, an unbiased approach for theidentification of kinase substrates within complex protein mixtures.The KESTREL method previously has proven to be a powerful tool toidentify novel and physiological substrates for a diverse set of proteinkinases (17, 45–47). P90-hCALD1 represents one of a handful of puta-tive substrates detected in the screen reported here and is selectivelyphosphorylated by K-cyclin- but not cyclin D1-activated kinase.Caldesmon is a known substrate forCDK1, a kinase that is activated specif-

ically andselectivelyat theonsetofmitosis.Unexpectedly,we foundthatcyclinE- and cyclin A-activated CDK2 can also phosphorylate hCALD1. TheseCDK2complexes,whichareformedandactivatedincellsduringthe latestagesof the G1-phase, have not been implicated previously in caldesmon phospho-rylation. Although phosphorylation of CALD1 by these kinase complexes hasyet to be documented to arise in cells, our findings raise the possibility that awider range of cyclin-dependent kinasesmay affect the cytoskeleton bymod-

FIGURE 8. Phosphorylation of caldesmon in the presence of actin or calmodulin. A, effect of actin on hCALD1 phosphorylation. Recombinant GST-hCALD1 was incubated for 15min at 4 °C in the absence or presence of F-actin. Phosphorylation reactions were then performed using the indicated cyclin�CDK complexes and analyzed as described in the legendfor Fig. 5A. B, effect of calmodulin on hCALD1 phosphorylation. Recombinant GST-hCALD1 was incubated for 15 min at 4 °C in kinase buffer containing 2.5 mM CaCl2 or CaM and 2 mM

CaCl2, and phosphorylation reactions were then performed as described in A.

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FIGURE 9. Effects of K-cyclin expression on actin cytoskeleton and cell shape. A, K-cyclin expression affects actin stress fibers and cell shape in mammalian cells. U2OS cells weretransiently transfected with K-cyclin or control plasmid and a plasmid encoding EGFP. After 24 h, cells were fixed and F-actin structures visualized using fluorescently labeledphalloidin. Representative photomicrographs depicting EGFP (left) and phalloidin fluorescence (right) of the same image are shown. B, K-cyclin mediated modulation of actin filamentappearance depends on CDK6 activity and caldesmon phosphorylation. U2OS cells were transfected with EGFP plasmid and expression plasmids as indicated. F-actin structures werevisualized using phalloidin as described in A. A minimum of 100 EGFP positive cells for each plasmid combination was scored as to the distribution and appearance of the phalloidinsignal. The percentage of cells with contiguous fibers (white bars) or fragmented fibers (black bars) is depicted. Error bars represent standard deviation between three independent

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ulationofcaldesmonactivity. Invitro, caldesmonisphosphorylatedbyadiversesetof kinases, suggesting that thisprotein is tied into, andcontrolledby, a com-plex web of signaling events. Several of these kinases, such as calmodulin-de-pendent kinase II and casein kinase II, phosphorylate residueswithin the ami-no-terminalmyosin binding region ofCALD1 and abolish its interactionwithmyosin (54, 55). In contrast, mitotic CDK1 and MAPK/ERK both modifyCALD1within the carboxyl-terminal actin-binding region and affect its asso-ciation with actin, although the impact of these kinases on CALD1 functionappears todiffer tosomeextend.Recentevidencesuggests that invitroMAPK/ERKphosphorylationdoesnot abolish the associationofCALD1withF-actin,although it disables the trans-filament linkage and thus filament bundling (70).In contrast, mitotic CDK1 leads to full dissociation of CALD1 from F-actinfilaments (48).Thefunctionalsignificanceof thesedifferences isnotclear,but itmaybeexplainedbythedifferentarrayofphosphorylationcatalyzedbythetwokinases.MAPK/ERK is known tophosphorylate porcineCALD1on two sites,corresponding to the human Ser-759 and Ser-789, both positioned at theextreme carboxyl terminus of the actin-binding region ofCALD1. In contrast,mitoticCDK1canphosphorylateadditionalsites, includingtwo(Thr-730,Thr-753)positionedadjacenttoamoreinternalactin-bindingsequence.Theresultsshown here reveal that K-cyclin�CDK6 phosphorylates Thr-730 and Thr-753and thus may mirror the impact of CDK1 rather than ERK/MAPKphosphorylation.Although similar, the sites reported to be phosphorylated by CDK1

and those determined by our work for K-cyclin�CDK6 are not identical,indicating that the two kinases differ in site preference and/or themodeof substrate recognition. Amajor difference relates to Ser-724 that doesnot apparently acts as acceptor for K-cyclin�CDK6 catalyzed phosphatetransfer. It is unlikely that Ser-724 phosphorylation has evaded detec-tion. Three different peptides, one generated from cell-derivedhCALD1 and two derived from recombinant hCALD1, span thesequence containing this serine, showing it to be in a nonphospho-rylated form. Previous work characterizing phosphorylation ofchicken CALD1 by mitotic CDK1 also suggested mutually exclusivephosphorylation of the sites corresponding, in the human sequence,to Thr-753 and Thr-759 (49). Our analysis of K-cyclin�CDK6-phos-phorylated hCALD1 however clearly documents the existence of apeptide that carries phosphate on both Thr-753 and Thr-759, indi-cating that phosphorylation of one site does not exclude phospho-rylation at the other. At present, it is not clear whether phosphoryl-ation of specific sites fulfills discernable functions, and conclusionsas to whether functionally distinct effects may arise from the differ-ences in phosphorylation by K-cyclin�CDK6 and mitotic CDK1 arecurrently not possible.Our in vitro analysis indicated that phosphorylation by K-cyclin�CDK6

impacts hCALD1 functions in ways that are indistinguishable from cyclinB�CDK1. Activation of CDK1 arises specifically during mitosis, and CALD1phosphorylationbythiskinase is implicatedinthedisassemblyof themicrofila-ment at the beginning of prophase, providing for unhindered chromosomesegregation and cytokinesis. In addition, recent work has linked the activationof CDK1 adjacent to the cell membranes in the promotion of cell movementduring interphase (34). In contrast, K-cyclin expression and associated kinaseactivityisconstantthroughoutthecellcycle,andK-cyclin�CDK6complexesarepresent both in the nucleus and in the cytoplasm in KSHV-transformed cells(71). Furthermore, K-cyclin-activated kinases are known to evade regulatoryloops that restrain the activity of cellular cyclin�CDKs in response to extracel-lular signaling cues (3). Together, these observations indicate that phosphoryl-

ation of caldesmon by K-cyclin-activated kinases may not be confined to aparticular cell cycle position or signaling context but may arise throughout,leading to constitutive caldesmon hyperphosphorylation and consequentialimpairment ofmicrofilament organization.Several independent observations in KSHV-infected cells are consist-

ent with aberrant modulation of actin cytoskeleton functions. Humanvascular endothelial cells, which normally form cobblestone-like cellarrays, are known to respond to KSHV infection with a striking shapechange leading to narrow, light-refractive cell bodies, loss of cell junc-tions, and decreased substratum adhesion (72, 73); this is quite reminis-cent of the response that arises in the U2OS cells upon K-cyclin expres-sion, as shown above (72, 73). Thismorphological transformation is alsoreproduced in transgenic mice with endothelium selective K-cyclinexpression (74). The work presented here provides a possible molecularexplanation of how these morphological alterations are achieved.An apparently related question is why KSHV may have developed

means to affect microfilament structure and function. Disintegration ofthe actin cytoskeleton is known to occur upon infection with a widerange of animal viruses, including human herpesvirus 1 and 2 and thehuman immunodeficiency virus (75). In most instances, the means bywhich viruses achieve this effect have not been defined, but the wide-spread association of this responsewith viral infection suggests a benefitfor viral replication and/or virus spread.Importantly, there is strong evidence for alterations of the micro-

filament architecture during cancer development (76) and that mis-regulation of microfilament functions contributes to cancer invasion(77, 78). Several reports link loss of CALD1 function to oncogenesis(79). For instance, v-Src-transformed cells display a reduced expres-sion of CALD1 (80, 81), whereas v-ErbB2-transformed fibroblastsshow enhanced tyrosine phosphorylation of CALD1 that correlateswith stress fiber disassembly (82). Lastly, missplicing of the CALD1gene has been observed in glioma microvasculature and is associatedwith tight junction breakdown between endothelial cells and vascu-lar leakage (83). Thus, K-cyclin-induced phosphorylation of CALD1could provide a cancer-promoting event, independent of, and inaddition to, the impact of this cyclin on cell cycle progression andproliferation.

Acknowledgments—We thank the Post Genomics and Molecular InteractionsCentre, University of Dundee, for the mass spectrometry facilities, Dr. DavidCampbell for assistance with protein sequencing and phosphoamino acidanalysis, and Dr. Fumio Matsumura for making available the CALD1 7thexpression construct. Phospho-specific antibodies were purified and charac-terized by the staff of the Division of Signal Transduction Therapy, School ofLife Sciences, University of Dundee.

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Caldesmon Phosphorylation by K-cyclin�CDK6

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Sibylle MittnachtMaria Emanuela Cuomo, Axel Knebel, Georgina Platt, Nick Morrice, Philip Cohen and

Virus-cyclin·CDK6 Phosphorylation of CaldesmonRegulation of Microfilament Organization by Kaposi Sarcoma-associated Herpes

doi: 10.1074/jbc.M503877200 originally published online August 22, 20052005, 280:35844-35858.J. Biol. Chem. 

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