3312 Research Article

IntroductionPost-translational modification of proteins by SUMO regulates

central cellular processes including transcription, cell-cycle

regulation and DNA-damage response (Gill, 2004; Hay, 2005;

Muller et al., 2004; Watts, 2007). It seems that SUMOylation

regulates these distinct processes via modulation of specific protein-

protein interactions (Geiss-Friedlander and Melchior, 2007). In

vertebrates, three SUMO isoforms are expressed. SUMO1 shares

43% identity with SUMO2 and SUMO3, whereas the latter two are

closely related (sharing 97% identity) and are therefore collectively

referred to as SUMO2/3. Paralog-specific modification has been

reported for various substrates (Vertegaal et al., 2006). SUMO

proteins are covalently attached to their targets via an isopeptide

bond with the ε-amino group of a lysine residue. The conjugation

process involves an enzymatic cascade comprising the E1-activating

enzyme Aos1 (also known as Uba2 and Sae1), the E2-conjugating

enzyme Ubc9, and additional E3 ligases including protein inhibitor

of activated STAT (PIAS) family members, Ran-binding protein 2

(RanBP2) and human polycomb protein 2 (hPC2; also known as

PC2 and CBX4) (Anckar and Sistonen, 2007; Johnson, 2004). De-

SUMOylation is mediated by the action of SUMO-specific

isopeptidases that belong to the family of ubiquitin-like proteases

[Ulps; also known as sentrin-specific proteases (SENPs)] (Hay,

2007). Notably, in most cases only a small fraction of a given target

protein is SUMOylated, making it difficult to identify novel SUMO

substrates and elucidate the functional consequence of the

modification (Geiss-Friedlander and Melchior, 2007).

Centrin proteins are highly conserved from yeast to humans and

regulate various cellular functions (Bornens and Azimzadeh, 2007;

Pereira and Schiebel, 2001; Salisbury, 2007). Humans express at

least three centrin isoforms. Centrin-1 is expressed exclusively in

male germ cells, certain neurons and ciliated cells. Centrin-2 and

centrin-3 are ubiquitously expressed, and both localize to the

centrioles and the pericentriolar material (Salisbury, 2007). Centrin-

2 has been implicated in centriole duplication (Salisbury et al., 2002)

but this view has recently been challenged (Kleylein-Sohn et al.,

2007). Remarkably, ~90% of the centrin-2 protein is not associated

with centrosomal structures (Paoletti et al., 1996) and instead much

of it is present within the nucleus. In line with this localization,

centrin-2 has been implicated in mRNA export from the nucleus

(Resendes et al., 2008) and this function seems to be conserved

(Fischer et al., 2004). Furthermore, within the nucleus, centrin-2

associates with the xeroderma pigmentosum group C (XPC) protein

and the human homolog of Rad23 B (HR23B) to form the trimeric

XPC complex (Araki et al., 2001). This complex functions as a key

component of the nucleotide excision repair (NER) pathway. It

detects and binds to DNA-damage sites in a reaction that requires

all three subunits (Nishi et al., 2005; Sugasawa et al., 1996), thus

representing a first line of defense against carcinogenesis (Batty

and Wood, 2000; de Laat et al., 1999; Sugasawa and Hanaoka, 2007;

Thoma and Vasquez, 2003). Intriguingly, the XPC protein becomes

modified by ubiquitin and SUMO in response to DNA damage

(Sugasawa et al., 2005; Wang et al., 2007; Wang et al., 2005), and

this has been shown to depend on an intact proteasome system

(Wang et al., 2005). Modification of the XPC protein was reported

to alter the DNA-binding properties of the complex and to be

essential for efficient NER (Sugasawa et al., 2005; Wang et al.,

2007).

Here we identify centrin-1 and centrin-2 as novel substrates of

SUMO. We demonstrate that SUMOylation regulates centrin-2

localization to the nucleus and we identify the SUMO E3-like ligase

hPC2 and SUMO2/3 (but not SUMO1) to be involved in this

process. Centrin-2 SUMOylation occurs independently of factors

that regulate SUMO modification of its binding partner, the XPC

protein. Yet, cells depleted of the XPC protein phenocopy the

mislocalization of centrin-2 that is seen upon inhibition of the

Centrins are multifunctional Ca2+-binding proteins that are

highly conserved from yeast to humans. Centrin-2 is a core

component of the centrosome of higher eukaryotes. In addition,

it is present within the nucleus, in which it is part of the

xeroderma pigmentosum group C (XPC) complex, which

controls nucleotide excision repair (NER). Regulation of the

subcellular distribution of centrin-2 has so far remained elusive.

Here we show that centrin-2 is a substrate of SUMOylation in

vitro and in vivo, and that it is preferentially modified by

SUMO2/3. Moreover, we identify the SUMO E3-like ligase

human polycomb protein 2 (PC2; also known as hPC2) as

essential for centrin-2 modification. Interference with the

SUMOylation pathway leads to a striking defect in nuclear

localization of centrin-2 and accumulation in the cytoplasm,

whereas centrosomal recruitment of centrin-2 is unaffected.

Depletion of the XPC protein mimics this situation and we

provide evidence that SUMO conjugation of centrin-2 enhances

its binding to the XPC protein. These data show that the

nucleocytoplasmic shuttling of centrin-2 depends on the SUMO

system and indicates that localization of centrin-2 within the

nucleus depends on its ability to bind to the XPC protein.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/18/3312/DC1

Key words: Centrins, Centrin-2, XPC, SUMO, hPC2,

Nucleocytoplasmic shuttling

Summary

SUMO-dependent regulation of centrin-2Ulf R. Klein1,* and Erich A. Nigg1,2

1Department of Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany2Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland*Author for correspondence (uklein@biochem.mpg.de)

Accepted 15 June 2009Journal of Cell Science 122, 3312-3321 Published by The Company of Biologists 2009doi:10.1242/jcs.050245

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3313Regulation of centrin-2

SUMOylation system and we show that the SUMOylation status

of centrin-2 influences its binding to the XPC protein. We conclude

that the nucleocytoplasmic shuttling of centrin-2 as well as its

binding to the XPC protein are regulated by the SUMO system.

ResultsCentrin proteins are modified by SUMO in vitro and in vivoPreliminary data obtained in our laboratory suggested that

centrin-2 might be a target of SUMOylation (our unpublished

observations). To explore this possibility, we first used yeast two-

hybrid assays to monitor binding of the essential SUMO E2-

conjugating enzyme Ubc9 to the three human centrin isoforms.

Interestingly, all centrin proteins showed binding to Ubc9, whereas

no binding between the control protein INCENP and Ubc9 was

observed (Fig. 1A). Additionally, centrin-1 and centrin-2 preferential

bound to SUMO2 in this assay, whereas centrin-3 did not interact

with the SUMO isoforms (Fig. 1B). Next, we analyzed centrin

modification by SUMO in an in vitro SUMOylation assay. We

detected conjugation of centrin-1 and centrin-2, but not centrin-3,

to SUMO3 (Fig. 1C). In order to analyze SUMO conjugation in

vivo we overexpressed the Myc-tagged centrin isoforms in

combination with His-tagged SUMO2. SUMOylation of centrins

was monitored by nickel nitrilotriacetate (Ni-NTA) pulldown of His-

SUMO2 conjugates followed by anti-Myc immunoblotting. In

agreement with our in vitro SUMOylation assay, only centrin-1 and

centrin-2 but not centrin-3 were present in the His-SUMO2

pulldown fractions (Fig. 1D). These data identify centrin proteins,

in particular centrin-1 and centrin-2, as novel targets of SUMO

modification.

Centrin-2 localization to the nucleus (but not to thecentrosome) depends on the SUMOylation systemBecause centrin-1 is not expressed ubiquitously, we focused our

further studies on the elucidation of the mechanism and function

of centrin-2 SUMOylation. To analyze a potential effect of the

SUMO system on centrin-2 localization to the centrosome, we first

depleted the essential SUMO E2-conjugating enzyme Ubc9 by

siRNA. Effective knockdown was verified by western blotting (Fig.

2A). We then used immunofluorescence microscopy to analyze

centrin-2 localization in cells that had been pre-extracted with 0.3%

Triton X-100 prior to methanol fixation. Under these conditions,

centrin-2 localization to the centrosome was not detectably impaired

by the depletion of Ubc9 (Fig. 2B). By striking contrast, we noticed

that the nuclear pool of centrin-2 was less abundant in Ubc9-depleted

cells than in GL2-treated controls (Fig. 2B). Because the total levels

of centrin-2 protein were unaffected by Ubc9 depletion (see Fig.

2A), it appeared likely that, upon Ubc9 knockdown, nuclear centrin-

2 was directed to the cytoplasm and was then extracted during

sample preparation. To substantiate this conclusion, we analyzed

nuclear centrin-2 in methanol-fixed cells that had not undergone

Fig. 1. Centrin proteins interact withcomponents of the SUMO system and aremodified by SUMO in vitro and in vivo.(A) Interaction of centrin-1, centrin-2 andcentrin-3 with the SUMO E2-conjugatingenzyme Ubc9 in directed yeast two-hybridassays. –LW indicates plates lackingleucine and tryptophan (non-selectivemedium), whereas QDO indicates plateslacking leucine, tryptophan, histidine andadenine (selective medium). All testedproteins were non self-activating. AD,activation domain; BD, binding domain.(B) Interaction of centrin-1 and centrin-2with SUMO2 but not SUMO1 in directedyeast two-hybrid assays. Tested proteinswere non self-activating. (C) Centrin-1 andcentrin-2 but not centrin-3 are modified bySUMO3 in vitro. 35S-labeled centrinproteins, generated by in vitro transcriptionand translation (IVT), were incubated withrecombinant E1, E2 and SUMO3 in thepresence of ATP. In control reactions the E1enzyme was omitted. Centrin-SUMOconjugates are indicated by an arrow.(D) Myc-tagged centrin proteins or theempty Myc-vector and His-tagged SUMO2were coexpressed in COS-7 cells. His-SUMO2 conjugates were recovered on Ni-NTA beads and subjected to westernblotting using anti-Myc antibody.Consistent with the in vitro data (C),centrin-1 and centrin-2 but not centrin-3 aremodified (marked by arrow). PD,pulldown.

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any pre-extraction step. Under these conditions, centrin-2 could

clearly be visualized in the cytoplasm of Ubc9-depleted cells,

whereas it was mostly absent from the nucleus (Fig. 2C).

Quantitative analysis revealed that 84.4% of total centrin-2 could

be detected in the nucleus in control depleted cells compared with

only 25.8% in Ubc9-depleted cells (Fig. 2D). Of note, the centrin-

2 interaction partner in the nucleus, the XPC protein, has been shown

to be modified by SUMO (Wang et al., 2007; Wang et al., 2005).

However, in contrast to centrin-2, Ubc9 depletion did not alter the

localization of the XPC protein to the nucleus (89.4% nuclear

localization in control depleted cells versus 89.2% in Ubc9

knockdown) (see Fig. 2D,E). Additionally, depletion of Ubc9 did

not detectably affect the nucleocytoplasmic distribution of centrin-

3 (Fig. 2F).

To analyze endogenous SUMOylation of centrin-2 and address

the possibility of a paralog-specific modification in vivo, we

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performed immunoprecipitation experiments with either anti-

SUMO1 or anti-SUMO2/3 antibodies. We detected a single anti-

centrin-2-reactive band indicative of mono-SUMOylation in

SUMO2/3 but not in SUMO1 immunoprecipitates (Fig. 3A).

Conversely, modified RanGAP1 was found in SUMO1

immunoprecipitates, whereas XPC did not show SUMO

modification under these conditions (Fig. 3A). We attempted to

exploit this difference to investigate a paralog-specific role for

SUMOylation in the in vivo regulation of centrin-2 localization. To

this end, we depleted either SUMO1 or SUMO2/3 by specific

siRNA duplexes. Western blotting showed effective knockdown of

SUMO1 or SUMO2/3 conjugates (Fig. 3B). Depletion of SUMO2/3

resulted in a strongly reduced nuclear localization of centrin-2 and

a concomitant accumulation of centrin-2 in the cytoplasm (Fig. 3C,

bottom row), in line with the effect of Ubc9 knockdown (Fig. 2C).

By contrast, no effect on centrin-2 localization was observed in

Fig. 2. The SUMO system regulatesnuclear localization of centrin-2. (A) HeLacells were treated with siRNA duplexesspecific for GL2 or Ubc9 and incubated for72 hours. Lysates were probed with theindicated antibodies by western blotting. α-tubulin serves as a loading control.(B) Experiment as in A but cells were fixed(as indicated in the text) and analyzed byimmunofluorescence with anti-centrin-2and anti-γ-tubulin antibodies. Note thereduced amount of centrin-2 in the nucleusof Ubc9-depleted (siUbc9) cells.(C) Experiment analyzing centrin-2 nuclearlocalization as in B except that the pre-extraction step during sample preparationwas omitted (see text). Note that centrin-2clearly fails to localize to the nucleus andinstead accumulates in the cytoplasm.(D) Quantification of centrin-2 and XPCsubcellular localization in control depletedversus Ubc9-depleted cells. The amount ofnuclear and cytoplasmic fluorescence isgiven as the percentage of total centrin-2fluorescence or XPC fluorescence. n=30cells per condition for centrin-2; n=20 cellsper condition for XPC. (E) Experiment asin C but analyzing XPC protein.(F) Experiment as in C but analyzingcentrin-3. DNA was stained with DAPI.Scale bars: 10 μm.

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3315Regulation of centrin-2

response to depletion of SUMO1 (Fig. 3C, middle row). This

indicates that centrin-2 localization to the nucleus requires an intact

SUMO system and, in particular, SUMO2/3.

The SUMO E3-like ligase hPC2 is essential for theSUMOylation of centrin-2To identify factors that regulate centrin-2 modification by SUMO,

we analyzed binding of centrin-2 to candidate SUMO E3 ligases

in the yeast two-hybrid assay. Among the tested candidates, only

hPC2 showed interaction with centrin-2 (Fig. 4A). Interestingly,

hPC2 is associated with Polycomb bodies within the nucleus but

only few SUMO substrates have previously been identified to

undergo hPC2-mediated modification (Wotton and Merrill, 2007).

To address a putative SUMO E3-ligase function of hPC2 towards

centrin-2, in vitro translated Myc-tagged hPC2 and the Myc-tagged

catalytic fragment of the control E3 ligase RanBP2 (RanBP2�FG)

were added to an in vitro SUMOylation reaction on centrin-2. The

assay was performed under limiting E1 and E2 concentrations,

because high concentrations can bypass the need for E3 ligases in

this set-up. Under these conditions, basal E1-E2-mediated centrin-

2 SUMOylation was weak but significantly enhanced in the reaction

containing hPC2 (Fig. 4B, compare lanes 2 and 3). The addition

of RanBP2�FG did not change the level of centrin-2 SUMOylation

(Fig. 4B, compare lanes 2 and 4). In order to address the requirement

of hPC2 for the SUMOylation of centrin-2 in vivo, cells expressing

Myc-tagged centrin-2 and His-tagged SUMO2 were depleted of

hPC2 or control proteins. Knockdown of transfected Myc-tagged

hPC2 was reproducibly observed with the siRNA duplexes used

(Fig. 4C). SUMOylation of centrin-2 was monitored by Ni-NTA

pulldown of His-SUMO2 conjugates followed by immunoblotting

with anti-Myc antibodies (Fig. 4D). Importantly, depletion of hPC2

resulted in a loss of centrin-2 SUMOylation similar to the result

obtained after knockdown of Ubc9. By contrast and attesting to the

specificity of this assay, centrin-2 SUMOylation was unaltered when

cells were depleted of the α- and β-forms of the control E3 ligase

PIAS2 (Fig. 4D). Immunofluorescence analysis of hPC2-depleted

cells showed a defect in nuclear targeting of centrin-2 (Fig. 4E),

which exactly mirrors the phenotype seen in cells depleted of Ubc9

and SUMO2/3 (see Fig. 2C; Fig. 3C). Taken together, these data

suggest that hPC2 acts as a SUMO E3 ligase towards centrin-2.

SUMOylation of centrin-2 occurs independently of factors thatregulate modification of the XPC proteinThe binding partner of centrin-2 within the nucleus, the XPC protein,

has been reported to be modified by ubiquitylation and

SUMOylation. These modifications were shown to occur in

response to ultraviolet (UV) irradiation (Sugasawa et al., 2005;

Wang et al., 2005) and to require an intact proteasome system,

because treatment with the proteasome inhibitor MG132 abolishes

them (Wang et al., 2005). We therefore investigated whether UV

irradiation and/or treatment with MG132 influence centrin-2

SUMOylation. Cells expressing Myc-tagged centrin-2 and His-

tagged SUMO2 were either UV irradiated with 50 J/m2 and

incubated for an additional 30 minutes or treated with MG132 at

20 μM for 1 hour before lysate preparation. In agreement with

published data (Sugasawa et al., 2005; Wang et al., 2005), we

observed the characteristic upshift of the XPC protein after UV

irradiation. Ni-NTA pulldown of His-SUMO2 conjugates revealed

that neither UV irradiation nor MG132 treatment detectably

influence the SUMOylation status of centrin-2 (Fig. 5A). In

addition, immunofluorescence analysis showed no significant effect

on overall nuclear localization of either centrin-2 or XPC protein

under conditions of proteasome inhibition (Fig. 5B) or UV

irradiation (Fig. 5C).

SUMO modification of centrin-2 regulates its binding to theXPC proteinThe observation that both the XPC protein and centrin-2 are targets

of SUMOylation prompted us to analyze their mutual

Fig. 3. SUMO-paralog-specific influence on centrin-2 nuclear localization.(A) Immunoprecipitations were performed from total HeLa cell lysates usinganti-SUMO1- or anti-SUMO2/3-specific antibodies and immunoprecipitates(IP) were probed by western blotting with the indicated antibodies. Note thatcentrin-2 is conjugated to SUMO2/3; RanGAP1 serves as a control forSUMO1 conjugation. (B) HeLa cells were treated with siRNA duplexesspecific for GL2, SUMO1 or SUMO2/3 and incubated for 72 hours. TheSUMO2/3 oligonucleotide targets SUMO2 and SUMO3. Lysates were probedwith the indicated antibodies by western blotting. α-tubulin serves as a loadingcontrol. (C) Experiment as in B but cells were fixed and analyzed byimmunofluorescence with anti-centrin-2 antibodies. DNA was stained withDAPI. Scale bar: 10 μm.

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interdependence with regards to this modification. Of note, siRNA-

mediated depletion of either one of these proteins did not effect the

abundance of the other (Fig. 6A), confirming and extending

previous data (Nishi et al., 2005). We first analyzed the influence

of centrin-2 knockdown on XPC-protein modification and

localization. Consistent with the data shown above, UV irradiation

resulted in modification of the XPC protein. In agreement with

previous reports indicating that Ubc9 and modification of XPC at

the SUMO consensus site K655 was responsible for the

characteristic XPC upshift (Wang et al., 2007; Wang et al., 2005),

the higher-molecular-weight bands reactive with anti-XPC

antibodies were largely lost upon knockdown of Ubc9 (Fig. 6B).

By contrast, centrin-2 depletion did not detectably interfere with

XPC-protein modification generated by UV irradiation (Fig. 6B).

Journal of Cell Science 122 (18)

Moreover, the overall nuclear localization of XPC protein was

independent of centrin-2 (Fig. 6C). Next, we analyzed centrin-2

modification and localization under conditions of XPC-protein

depletion, again using Ubc9 depletion for control. XPC-protein

knockdown did not significantly influence either the SUMOylation

of centrin-2 (Fig. 6D) or its centrosomal targeting (data not shown).

Strikingly, however, in XPC-protein-depleted cells, centrin-2 no

longer localized efficiently to the nucleus and instead was found

dispersed in the cytoplasm (Fig. 6E). Depletion of XPA did not

affect centrin-2 targeting to the nucleus, ruling out that the observed

defect is due to inhibition of NER (Fig. 6F,G). Importantly, the

localization pattern seen after XPC depletion was identical to the

one observed after interference with the SUMOylation pathway (see

Figs 2-4). This raised the issue of whether unmodified centrin-2

Fig. 4. hPC2 is essential for SUMOylation and nuclear localization of centrin-2. (A) hPC2 binds to centrin-2. Centrin-2 was tested for interaction with SUMO E3ligases in directed yeast two-hybrid assays as described in Fig. 1. AD, activation domain; BD, binding domain. (B) In vitro SUMOylation assay as outlined in Fig.1, performed under limiting E1 and E2 concentrations. Untagged centrin-2 was incubated in a SUMOylation reaction in the presence of the indicated Myc-taggedSUMO E3 ligases. Anti-Myc western blotting serves as a loading control for the SUMO E3 ligases. Note enhanced SUMOylation of centrin-2 in the reactioncontaining hPC2 (compare lane 3 to lanes 2 and 4). Arrow indicates centrin-2–SUMO conjugate. (C) HeLa cells overexpressing Myc-tagged hPC2 were incubatedwith siRNA duplexes specific for GL2 or hPC2 and incubated for 72 hours. Lysates were probed with the indicated antibodies by western blotting. α-tubulin servesas a loading control. (D) hPC2 is required for SUMOylation of centrin-2 in vivo. Myc-tagged centrin-2 and either HA- or His-tagged SUMO2 were coexpressed inHeLa cells treated with siRNA oligonucleotides directed against the indicated proteins. The siRNA directed against PIAS2 targets the α- and β-isoforms. His-SUMO2 conjugates were recovered on Ni-NTA beads and western blotting was performed with the indicated antibodies. The asterisk denotes a PIAS2 cross-reactive band. (E) HeLa cells were treated with siRNA duplexes specific for GL2 or hPC2 and incubated for 72 hours. Immunofluorescence was performed withanti-centrin-2 antibodies. DNA was stained with DAPI. Scale bar: 10 μm.

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3317Regulation of centrin-2

fails to localize to the nucleus because of an import defect or,

alternatively, because of an inability to bind to nuclear XPC

protein. To determine whether SUMOylation of centrin-2 might

directly regulate its binding to the XPC protein (and in consequence

its nuclear localization), we set up an in vitro binding assay. As

shown in Fig. 7A, we combined in vitro transcription and translation

with in vitro SUMOylation to prepare Myc-tagged centrin-2 in both

a modified and an unmodified form. For control, borealin, another

SUMOylation substrate (Klein et al., 2009), was similarly prepared.

Immunoprecipitations were carried out with anti-Myc or control

antibodies, and reactions were subsequently incubated with in-vitro-

translated untagged XPC protein. Samples were analyzed by

autoradiography (to detect 35S-labeled centrin-2 and borealin) and

probed by western blotting with anti-XPC antibodies for co-

precipitation of the XPC protein (Fig. 7B). Anti-SUMO2/3 western

blotting was used to confirm SUMO2/3 conjugates. No binding of

XPC protein could be seen in immunoprecipitates prepared with

control antibodies (Fig. 7B, lane 2) or in anti-Myc

immunoprecipitates containing SUMOylated borealin (Fig. 7B, lane

5). Only a modest fraction of XPC protein was found in the

immunoprecipitation of unmodified centrin-2, but a striking increase

in binding of the XPC protein was observed in immunoprecipitations

of SUMOylated centrin-2 (Fig. 7B, compare lanes 3 and 4). To

strengthen this finding, we performed centrin-2

immunoprecipitations from control depleted cells and cells that had

been depleted of Ubc9. In agreement with the in vitro assay

described above, the amount of XPC protein co-precipitating with

centrin-2 was substantially higher in control depleted cells compared

with cells in which SUMOylation was blocked by Ubc9 knockdown

(Fig. 7C, compare lanes 5 and 6). These results suggest that the

SUMOylation of centrin-2 enhances its binding to the XPC protein,

which in turn is required for efficient accumulation of centrin-2

within the nucleus.

DiscussionWe report the identification of centrin-1 and centrin-2 as novel

substrates of post-translational modification by SUMO. Within the

human centrin protein family, centrin-1 and centrin-2 are most

closely related (83.7%), making it likely that a common structural

feature mediates SUMO conjugation to these two proteins. By

contrast, we did not observe SUMOylation of centrin-3, which is

only 51.7% identical to centrin-1 and 52.3% identical to centrin-2.

Of note, centrin-3 is the closest homolog of Cdc31p (51.4%

identity), the single centrin protein expressed in the yeast

Saccharomyces cerevisiae. Thus, it should not necessarily be

expected that centrin SUMOylation represents a conserved

mechanism that also regulates Cdc31p in yeast.

In this study, we have focused on the mechanism and function

of SUMO conjugation to centrin-2. Although we have not observed

any obvious impact of SUMOylation on the centrosome association

of centrin-2, we show that the SUMO system is required for efficient

centrin-2 localization to the nucleus, in which this protein is

implicated in the NER pathway (Nishi et al., 2005). In particular,

we demonstrate that the SUMO E3-like ligase hPC2 is essential

for centrin-2 modification and, accordingly, its nuclear localization.

hPC2 localizes to Polycomb bodies within the nucleus but its role

as a SUMO E3 ligase was recognized only recently (Kagey et al.,

2003). A subsequent study has shown that the E3-ligase activity of

Fig. 5. Centrin-2 SUMOylation occurs independentlyof UV irradiation and the proteasome system.(A) HeLa cells expressing Myc-tagged centrin-2 andeither HA- or His-tagged SUMO2 were either leftuntreated, irradiated with UV (50 J/m2) andincubated for an additional 30 minutes or treated withthe proteasome inhibitor MG132 (20 μM) for 1 hour.His-SUMO2 conjugates were recovered on Ni-NTAbeads and western blotting was performed with theindicated antibodies. (B) HeLa cells were incubatedwith DMSO or MG132 (20 μM) for 1 hour andanalyzed by immunofluorescence using anti-centrin-2 and anti-XPC-protein antibodies. (C) HeLa cellswere irradiated with UV (50 J/m2), incubated for anadditional 30 minutes and fixed.Immunofluorescence was performed with antibodiesdirected against centrin-2, XPC protein and γH2AXas a marker for DNA damage. DNA was stained withDAPI. Scale bar: 10 μm.

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hPC2 is not linked to its localization to Polycomb bodies (Kagey

et al., 2005). Hence, hPC2-mediated SUMOylation of centrin-2

could well take place outside of Polycomb bodies. It has been

speculated that hPC2 might be recruited to DNA via interaction

with a DNA-binding protein in order to SUMOylate regulators of

transcription (Kagey et al., 2005). In this context, it is intriguing

that centrin-2 associates with DNA as part of the XPC complex

(Araki et al., 2001), and we show in this study that hPC2 interacts

with centrin-2. Thus, it will be interesting to explore whether centrin-

2 in turn influences the localization and/or function of hPC2.

Our data suggest a paralog-specific modification of centrin-2. In

fact, SUMO2/3 (but not SUMO1) showed interaction with centrin-

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2, and depletion of SUMO2/3 (but not SUMO1) influenced centrin-

2 localization to the nucleus. Analysis of the conserved lysine

residues present in the related SUMO substrates centrin-1 and

centrin-2 reveals an extensive overlap (19 out of the total 25 lysine

residues present in the centrin-2 sequence, with only seven of these

not present in the non-SUMO substrate centrin-3), making it

difficult to predict the specific lysine residue that might serve as

the SUMO acceptor site (supplementary material Fig. S1A).

Interestingly, mutation of the five SUMO consensus sites present

within centrin-2 did not inhibit SUMOylation substantially

(supplementary material Fig. S1B,C). In view of a recent proteomic

screen showing that more than half of all SUMO2/3 substrates

Fig. 6. XPC protein localizes centrin-2 to thenucleus. (A) HeLa cells were incubated for 72hours with siRNA duplexes specific for GL2,centrin-2 or XPC protein. Cell lysates were probedwith the indicated antibodies by western blotting.α-tubulin serves as a loading control. (B) HeLacells were incubated for 72 hours with siRNAduplexes specific for GL2, Ubc9 or centrin-2,irradiated with UV (50 J/m2) and incubated for anadditional 30 minutes. Cell lysates were probedwith the indicated antibodies by western blotting.(C) HeLa cells were treated for 72 hours withsiRNA duplexes specific for GL2, centrin-2 orXPC protein, fixed and analyzed byimmunofluorescence using anti-XPC-proteinantibodies. Depletion of centrin-2 or XPC proteinresults in disrupted cell morphology. (D) Myc-tagged centrin-2 and either HA- or His-taggedSUMO2 were coexpressed in HeLa cells treatedwith siRNA oligonucleotides directed against theindicated proteins. His-SUMO2 conjugates wererecovered on Ni-NTA beads and western blottingwas performed with the indicated antibodies.(E) Depletion of XPC protein interferes withnuclear localization of centrin-2. Experiment as inC analyzing centrin-2 localization. (F) HeLa cellswere incubated for 72 hours with siRNA duplexesspecific for GL2 or XPA protein. Cell lysates wereprobed with the indicated antibodies by westernblotting. (G) Experiment as in E under controlconditions or XPA depletion. DNA was stainedwith DAPI. Scale bar: 10 μm.

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3319Regulation of centrin-2

identified lacked the established Kx[E/D] consensus motif (Blomster

et al., 2009), this is perhaps not surprising. Thus, given the

multitude of lysines in centrin-2, it might be difficult to interfere

with SUMOylation through specific mutagenesis of this protein.

Nevertheless, the observation that depletion of Ubc9, hPC2 or

SUMO2/3 (all demonstrated to interact with centrin-2) all abolish

centrin-2 localization to the nucleus strongly suggests that

SUMOylation of centrin-2 (rather than SUMOylation of another

substrate) is directly responsible for its nuclear localization. Our

data indicate that a non-SUMOylatable centrin-2 mutant will

exhibit defects in XPC-protein binding and targeting to the nucleus.

XPC protein, the nuclear centrin-2 interaction partner, is

conjugated to ubiquitin and SUMO in response to UV irradiation

(Sugasawa et al., 2005; Wang et al., 2007; Wang et al., 2005), and

this has been shown to be regulated by the proteasome system (Wang

et al., 2005). In yeast, Rad33p was identified recently as a binding

partner of Rad4 (the yeast homolog of the XPC protein) and was

shown to regulate modification of Rad4 protein under conditions

of DNA damage, leading to the speculation that yeast Rad33p might

function similarly to mammalian centrin (den Dulk et al., 2008).

Here, we demonstrate that SUMOylation of centrin-2 occurs

independently of UV irradiation and the proteasome system.

Moreover, in contrast to data from yeast (den Dulk et al., 2008),

centrin-2 depletion did not influence XPC-protein modification in

HeLa cells. We thus conclude that the post-translational

modifications of the XPC protein and centrin-2 depend on different

factors.

It is striking that SUMO modification of two different subunits

of the XPC complex contributes to its regulation. Although

SUMOylation of the XPC protein was shown to be required for the

recruitment of XPG to sites of DNA damage and regulate the NER

efficiency of the complex (Wang et al., 2007), we show here that

SUMO modification of centrin-2 is required for efficient interaction

with the XPC protein, which in turn seems to result in the

accumulation of centrin-2 within the nucleus. However, we also

observed binding (albeit to a much lesser extent) of non-

SUMOylated centrin-2 to XPC protein, in agreement with previous

in vitro binding studies (Araki et al., 2001) and our notion that

nuclear localization of centrin-2 was not completely abolished upon

interference with the SUMOylation pathway. Structural studies

using small peptides showed residues 847-863 of the XPC protein

to associate with centrin-2 in vitro (Popescu et al., 2003; Thompson

et al., 2006). However, these experiments did not address the post-

translational modifications on either XPC protein or centrin-2. Thus,

in future structural studies it will be interesting to evaluate binding

of the full-length XPC protein and centrin-2 under conditions in

Fig. 7. SUMOylation-dependent binding of centrin-2 to XPCprotein. (A) Preparation of proteins to be used for in vitro co-immunoprecipitation experiments. In vitro transcribed andtranslated (IVT) Myc-tagged proteins were either modified bySUMO3 or left unmodified (SUMO reaction lacking the E1-activating enzyme). Asterisks indicate SUMO modification.(B) SUMOylation of centrin-2 enhances its binding to XPCprotein. Modified versus non-modified Myc-tagged centrin-2(and modified Myc-tagged borealin as control) wereimmunoprecipitated using anti-Myc antibodies. Subsequently,in vitro transcribed and translated untagged XPC protein wasadded to the immunoprecipitations and samples wereincubated in binding buffer for 2 hours at 4°C. Afterwards,samples were subjected to SDS-PAGE. ImmunoprecipitatedMyc-tagged proteins were monitored by autoradiography andbound XPC protein was detected by western blotting withanti-XPC antibodies. Anti-SUMO2/3 western blotting showsdetection of SUMO2/3 conjugates. Asterisks indicate SUMOmodification. The IgG immunoprecipitation and theSUMO2/3-modified Myc-tagged borealin protein serve asnegative controls. (C) Immunoprecipitations (IP) wereperformed with control IgG or anti-centrin-2 antibody fromcontrol or Ubc9-depleted total HeLa cell lysates. Input (IN)and pulldown fractions were probed with the indicatedantibodies by western blotting. Under conditions of Ubc9depletion, binding of XPC to centrin-2 is significantly reduced(compare lanes 5 and 6). Asterisk indicates the centrin-2–SUMO2/3 conjugate. Double asterisk denotesimmunoglobulins.

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3320 Journal of Cell Science 122 (18)

which the two proteins have undergone post-translational

modifications.

Cdc31p/centrin-2 are core components of the yeast spindle pole

body or the centrosome of higher eukaryotes, respectively. Although

little is presently known about a possible role of SUMO at the

centrosome, recent studies have identified several proteins that

undergo SUMOylation and are known to localize to both

centrosomal structures and the nuclear compartment (Cheng et al.,

2006; Haindl et al., 2008; Yun et al., 2008). Our present data suggest

that the SUMOylation machinery does not influence centrin-2

localization to the centrosome but is required for the efficient

accumulation of the protein in the nucleus, where it functions in

NER (Nishi et al., 2005) and mRNA export (Fischer et al., 2004;

Resendes et al., 2008). A priori, the nuclear accumulation of centrin-

2 could be regulated at the level of transport or retention (or both).

The small size of centrin-2 (~16 kDa) would allow for passive

diffusion across the nuclear membrane and, consistent with this,

the inhibition of Crm-1-mediated nuclear export by leptomycin-B

treatment did not change the localization of centrin-2 to the

centrosome (data not shown). Our data identifies SUMOylation-

dependent binding to the XPC protein as a crucial step for centrin-

2 nuclear recruitment. Thus, the equilibrium distribution of centrin-

2 between the cytoplasm and the nucleus is most probably

determined by its binding to interaction partners in the two

compartments. This interpretation is supported by the finding that

overexpression of the XPC protein leads to enhanced nuclear

accumulation of centrin-2 (Charbonnier et al., 2007).

Numerous centrosomal proteins are known to reside also within

the nucleus but their nuclear functions, if any, are unknown and the

regulation of their subcellular localization have not been addressed.

Extrapolating from the data reported here, we consider it attractive

to speculate that SUMOylation of centrosomal proteins might

represent a general mechanism to influence their nucleocytoplasmic

partitioning. Thus, the nucleus might conceivably serve as a

reservoir for proteins involved in centrosome function and the

regulation of their nucelocytoplasmic distribution could represent

an additional level of control. In support of this hypothesis, shuttling

of centrosomal proteins has recently been implicated in the process

of centrosome duplication (Prosser et al., 2009).

Materials and MethodsDirected yeast two-hybrid assayscDNAs encoding the respective prey or bait proteins were cloned in frame with the

GAL-activation domain of pACT2 or pGAD vectors or the GAL-binding domain of

pFBT9 or pGBD vectors and tested for self-activation. Directed yeast two-hybrid

assays were performed as described (Klein et al., 2006).

In vitro SUMOylationProteins were generated by in vitro transcription and translation in the presence of35S-labeled methionine using the TNT Quick Coupled T7 kit (Promega). Recombinant

SUMO1 and SUMO3 were purchased from LAE Biotech. SUMOylation was carried

out using the SUMOylation control kit according to the manufacturer’s instruction

(LAE Biotech). For Fig. 4B, lower concentrations of Aos1 (100 nM) and Ubc9 (70

nM) were used. To analyze SUMOylation-dependent binding of XPC protein to

centrin-2, Myc-tagged centrin-2 (or borealin) generated by in vitro transcription and

translation was in vitro SUMOylated with SUMO3 (minus E1-activating enzyme for

the control reaction) as outlined above and immunoprecipitated with anti-Myc

antibodies (9E10) or control IgGs. Immunoprecipitations were washed three times

in washing buffer [50 mM Tris/HCl, pH 7.4, 250 mM NaCl, 1 mM DTT, 0.3% NP40

and protease inhibitors (complete, Roche)] and incubated with in vitro transcribed

and translated untagged-XPC protein (35S-methionine omitted) in binding buffer [50

mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM DTT, 0.1% NP40 and protease inhibitors

(complete, Roche)] for 2 hours at 4°C. Subsequently, samples were washed three

times with binding buffer, boiled for 5 minutes at 95°C in 5� sample buffer and

analyzed by SDS-PAGE and subsequent western blotting (for XPC protein) or

autoradiography (for centrin-2).

In vivo SUMOylationFollowing transfection of SUMOylation targets and His-tagged SUMO constructs,HeLa S3 or COS-7 cells were incubated for 48 hours and Ni-NTA precipitations weredone as described (Muller et al., 2000). Where indicated, siRNA duplexes weretransfected 72 hours before lysate preparation and subsequent Ni-NTA precipitation.The protocol to monitor SUMOylation of endogenous proteins by SUMO pulldownhas been described before (Klein et al., 2009). For the experiment shown in Fig. 7C,the protocol was applied using 6 μg of control IgG or anti-centrin-2 antibody forimmunoprecipitation.

Immunofluorescence and immunoblottingMethanol fixation was performed according to standard protocols andimmunofluorescence was performed as described (Klein et al., 2006) with thefollowing primary antibodies: anti-γ-tubulin (Sigma), anti-centrin-2 (Kleylein-Sohnet al., 2007), anti-XPC (Santa Cruz Biotechnology) and anti-γH2AX (Millipore). Anti-centrin-3 antibody was a kind gift of Michel Bornens, Institut Curie, Paris, France.Secondary antibodies were Cy2/Cy3-conjugated donkey antibodies (Dianova). Forquantification, the cell body was visualized by α-tubulin (Sigma) staining and DAPIwas used to identify the nuclear compartment. Centrin-2 and XPC nuclear fluorescencewas defined as fluorescence overlap with DAPI staining. Cytoplasmic fluorescencewas calculated as the difference between total cell fluorescence and nuclearfluorescence. All analyses were performed using ImageJ software. The followingantibodies were used for western blotting: anti-SUMO1 (clone 21C7, Zymed), anti-SUMO2/3 (clone 1E7, MBL), anti-PIAS2 (clone 116, Sigma-Aldrich), anti-Ubc9(clone 50, BD Biosciences), anti-Myc (9E10), anti-centrin-2 (Kleylein-Sohn et al.,2007), anti-XPA (Santa Cruz Biotechnology), anti-XPC (GeneTex) and anti-α-tubulin(DMA1, Sigma-Aldrich). Anti-RanGAP1 was a kind gift of Frauke Melchior,University of Göttingen, Germany. Secondary antibodies conjugated to horseradishperoxidase were obtained from Jackson ImmunoResearch Laboratories. To avoid IgGchain signals in the anti-SUMO2/3 western blot shown in Fig. 7B, secondary anti-mouse TrueBlot (e-Bioscience) was used.

Cell culture, transfection and siRNA treatmentHeLaS3 or COS-7 cells were grown under standard conditions. Plasmid transfectionswere performed using FuGENE 6 reagent (Roche Diagnostics). mRNAs were targetedas follows: Ubc9 (Klein et al., 2009); SUMO1 (sc-29498A, Santa Cruz Biotechnology);SUMO2/3 (targeting SUMO2 and SUMO3), 5�-GUCAAUG AGG CA -GAUCAGAdTdT-3�; hPC2#1, 5�-CGUGGGAAC CGGAGGAGAAdTdT-3�;hPC2#2, 5�-GUUUGUACGUGGUGUUAUUdTdT-3� (a mixture of hPC2#1 andhPC2#2 was used to achieve efficient hPC2 knockdown); centrin-2, 5�-GCACAUGUAACUAGAUUUAdTdT-3�; XPC (Despras et al., 2007); XPA (SantaCruz Biotechnology); and PIAS2 (targeting the α- and β-isoforms) (Yang andSharrocks, 2005).

Treatment with MG132 and UV irradiationUV irradiation was performed using a CL-1000 UV-crosslinker (UVP, Upland, CA).HeLa S3 cells were irradiated with 50 J/m2, incubated for an additional 30 minutesand either subjected to the in vivo SUMOylation protocol (see above) or pelletedand directly dissolved in 2� sample buffer (Fig. 6B). HeLa S3 cells were treatedwith the proteasome inhibitor MG132 (Calbiochem) at a concentration of 20 μM for1 hour and subjected to the in vivo SUMOylation protocol (Fig. 5A) or processedfor immunofluorescence analysis (Fig. 5B).

We are grateful to Fumio Hanaoka for providing the anti-XPCantibody used for initial experiments, Michel Bornens for providinganti-centrin-3 antibody, Frauke Melchior for anti-RanGAP1 antibody,and the ‘centrosome lab’ for discussions, constructs and help. Wespecifically thank Julia Kleylein-Sohn and Stefan Müller for sharingunpublished data and Mikael LeClech for help. We also thank StefanMüller for critical reading of the manuscript. This work was supportedby the Max-Planck-Society and a fellowship from the BoehringerIngelheim Fonds to U.R.K.

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