Báo cáo khoa học: NBR1 interacts with fasciculation and elongation protein zeta-1 (FEZ1) and calcium and integrin binding protein (CIB) and shows developmentally restricted expression in the neural tube pptx

NBR1 interacts with fasciculation and elongation protein zeta-1 FEZ1 and calcium and integrin binding protein CIB and shows developmentally restricted expression in the neural tube Car

NBR1 interacts with fasciculation and elongation protein zeta-1 (FEZ1) and calcium and integrin binding protein (CIB) and shows

developmentally restricted expression in the neural tube

Caroline Whitehouse’, Julie Chambers’*, Kathy Howe", Martyn Cobourne?, Paul Sharpe”

and Ellen Solomon’

‘Division of Medical and Molecular Genetics, and *Department of Craniofacial Development, GKT School of Medicine,

Guy's Hospital, London, UK

NBRI (named as next to BRCA/) was originally cloned as a

candidate gene for the ovarian cancer antigen CA125, using

expression cloning with the anti-CA125 Ig, OC125 NBRI

has been of interest due to its position close to BRCA/,

although no involvement in breast or ovarian cancer has

been demonstrated Recently, the antigen CA125 has been

cloned, and identified as a new mucin, MUCI6, entirely

different from NBRI1 The function of NBR1 remains un-

known To investigate its function, a yeast two-hybrid study

was performed to identify interacting protein partners that

may reflect a biological role for this protein Here, we show

that NBRI interacts with two proteins; fasciculation and

elongation protein zeta-1 (FEZ1), a PKCC interacting pro-

tein, and calcium and integrin binding protein (CIB), which

is associated with polo-like kinases Fnk/Snk and _ the

Alzheimer’s disease presenilin 2 protein Co-transfection of FEZI and NBRI showed overlapping localization in the cytoplasm, whereas coexpression of NBR1 and CIB resulted

in a shift of CIB protein expression from the nucleus to the perinuclear compartment FEZ1 is highly expressed in the brain and in situ hybridization analysis of Nbr1 showed that its expression is also regulated in the murine brain during development These data suggest that NBR1 may function, through interaction with CIB and FEZ in cell signalling pathways, with a developmentally restricted expression suggesting a possible role in neural development

Keywords: ZZ zinc binding domain; OPR domain; UBA domain; CIB; FEZ]

The human NBRI gene (named for its location, next to

BRCAI]), originally named 1A1.3B, was cloned as a

candidate gene for the ovarian cancer antigen CA125 [1]

A serum assay for CA125 using the monoclonal antibody

OC125 showed that levels of this antigen are elevated in

> 80% of patients with epithelial ovarian cancer [2]

Recently a new mucin, MUCI6 has been cloned as

CA125 [3] whose biochemical characteristics as a high

molecular mass, heavily O-glycosylated protein correlate

Correspondence to C Whitehouse, Division of Medical and Molecular

Genetics, GKT School of Medicine, Guy’s Hospital, London SE1

ORT Fax: + 020 79558762, Tel.: + 020 79555000 ext 5585

E-mail: caroline.whitehouse@kcl.ac.uk

Abbreviations: NBR1I, next to BRCA1; CIB, calcium and integrin

binding protein; FEZ1, fasciculation and elongation protein zeta-1;

PKC ¢, protein kinase C zeta isoform; d.p.c., days post coitum; RBCC,

RING, B-box and coiled-coil protein; PML, promyelocytic leukaemia

gene; HA-, haemaglutinin epitope; EGFP, enhanced green fluorescent

protein; OPR, octicosapeptide sequence; UBA domain, ubiquitin-

associated domain; rfp, ret finger protein; BDH, acetic anhydride;

TESPA, 3-aminopropyltriethoxysilane

Note: further information on the proteins in this paper is available

from GenBank under the following accession numbers: NBR1

(X76952), CIB (U82226), FEZ1 (XM006241), PKCZ, (XM001533),

PML (XM007642)

* Present address Astra Zeneca, Alderley Park, Macclesfield, UK

(Received 24 September 2001, revised 14 November 2001, accepted 16

November 2001)

with the expected properties of the polypeptide carrying the CA125 epitope [4]

NBRI shares little homology with other known proteins, but does contain a number of protein motifs (see Fig 1A) These domains include two putative metal binding regions;

a zinc binding domain originally thought to belong to the B-box family of zinc binding domains and an octicosapep- tide sequence (OPR) also thought to be involved in divalent cation binding The octicosapeptide domain is a 28-residue motif also present in protein kinase C isoforms iota, lambda and zeta The B-box domain, however, more closely resembles the ZZ zinc-binding domain present in dystro- phin-like proteins, and CREB-bmding proteins/p300 homologues The ZZ domain in dystrophin is thought to bind calmodulin, and a missense mutation in one of the conserved cysteine residues in dystrophin was described in a patient with Duchene muscular dystrophy [5] NBR1 also contains a coiled-coil domain that is often implicated in protein-protein interactions [6] An ubiquitin-associated domain (UBA) has also been predicted at the C-terminus of NBRI [7] This domain is thought not to bind ubiquitin domains directly but is postulated to be involved in conferring target specificity to multiple enzymes of the ubiquitination system [8] The BRCAI protein has recently also been shown to be involved in the ubiquitination pathway, where cancer-associated mutations in the N-terminal RING finger domain of BRCA1 results in loss

of ubiquitin protein ligase (E3) activity [9] The related RBCC family of proteins which contain a RING finger domain in addition to a B-box and coiled-coil domain, and

A

Fig 1 Schematic representation of the protein (43.72 Ø11256 (288.329) 917-959

domains of NBR1 (A) (OPR) octicosapeptide ng =

repeat (ZZ) zinc finger (CC) coiled-coil and Ĩ $33 E3 |

(UBA) ubiquitin-associated domain The

amino acids encoding each domain are shown B

below and the figure is not drawn to scale

có pGBT9-NBRI constructs FEZI1 FEZI1 FEZI CIB pGAD424

(B) Yeast two-hybrid assay analysis identifies (214) (Y156) (Y163) (Y198) -NBRI CIB and FEZZl as Interacting partners of

NBRI HE7c cells were cotransformed with ) BUNS HÌ + aa aa aa er either pACT2FEZ]1 (Y214), pACT2FEZ1 i) BT

(Y 198) or pGAD424NBRI1 and

(1) pGBTONBRI1 Gi) pGBTINBR1 46 +++ +++ +†† +r† tt (iii) PGBTINBR1333 (iv) PGBTONBRI1eé term ) BT

(v) pGBTONBRIcl (v1) pGBTONBRI1c2

(ix) pGBKT7CIB and tested for protein—pro- vii) nH oc | + + + +

tein interaction by a colony lift B-galactosidase _

assay and growth on —leu-trp-his medium in |

(+ + +, strongly positive; + +, moderately pGBKT7CIB construct

positive; +, weakly positive; —, negative; n/d, » "- nid + not determined; MH, GAL4 BD)

includes members such as PML and the ret finger protein

(rfp), have been shown to have transforming activities when

inappropriately expressed [10]

The NBR1 protein is highly conserved, with 87% sequence

similarity with the murine homologue The murine Nbr/ gene

lies head to head with the Brcal gene, with the intragenic

region of 289 bp suggesting the possibility of co-ordinated

expression of the two genes Mutations in the human BRCA/

gene were found in 81% of breast—ovarian cancer families in

a large study [11] and are thought to be responsible for 45%

of cases of familial early onset breast cancer [12]

This study describes the results of a yeast two-hybrid

experiment to identify interacting partners of NBR1, and in

situ hybridization analysis of the murine Nbr] embryonic

expression pattern Two interacting partners were identified,

CIB and FEZ, which were also shown to interact directly,

and the interacting domains were delineated Nbr1 expres-

sion in the developing mouse embryo showed an early

uniform pattern of expression, which then becomes restric-

ted around 10.5—13.5 d.p.c (days post coitum) to the neural

tube, and then showed a wide expression pattern 1n the adult

mouse

MATERIALS AND METHODS

Yeast two-hybrid library screening

The full-length cDNA of human NBRI1 was cloned in frame

into the GAL4 DNA-binding domain (BD) vector pGBT9

(Clontech) The yeast strain HF7c was transformed with

this construct (pGBTONBRI1) and a human placenta

MATCHMAKER cDNA library fused with the GAL4

AD (Clontech) Transformants were selected on plates

lacking leucine, tryptophan and histidine, and containing

3 mm 3-amino-1,2,4-triazole (Sigma) for 5 days Putative

positive colonies were restreaked onto fresh master plates

for B-galactosidase assays These were performed following

colony-lifts onto Hybond-N nylon membranes and assayed

as described previously [13] The pACT2 library plasmid DNA from candidate clones was recovered and the cDNA inserts analysed by nucleotide sequencing to ensure the inserts were in frame with the GAL4 activation domain Any candidate clones were screened for false-positives by cotransformation with the empty pGBT9 vector or control plasmid pLAMS’ (Clontech)

Delineation of interacting regions Sections of the NBRI cDNA were PCR amplified and subcloned into the pGBT9 vector EcoRI site to give the following constructs: pGBTINBR1>,;¢ (amino acids 1—216), PGBTONBR1333 (amino acids 1-333), pGBIONBRIcl (amino acids 340-453), pGBITOYNBRIc2 (amino acids 451-597), pGBTONBRIc3 (amino acids 592-754) and pGBT9NBRIc4 (amino acids 748-910) A Bc/l fragment

of pGBTONBRI was subcloned into the BamHI site of pGBT9 to give a C-terminal construct, pGBTONBR1¢ tem (amino acids 494-849) All constructs were subsequently sequenced before testing in the yeast two-hybrid assay Interactions were scored as + + + (strongly positive), + + (moderately positive), + (weakly positive) or — (negative), where +++ denoted growth on —leu-trp-his plates in three days and a positive colony lift B-galactosidase assay 1n 4h A weakly positive result denoted growth on —leu- trp-his plates in five days and a positive colony lift B-galactosidase assay 1n 6 h

Northern blot analysis CIB and FEZ1 probes were excised from the pACT2 vector with BamHI/BgHl and EcoRI/Xhol, respectively The NBRI1 probe used for Northern blot analysis was as described previously [1] Human multiple tissue Northern blots (Clontech) were hybridized at 42 °C for 18-24 hin 5 x NaCl/P;/EDTA, 10 x Denhardts, 2% SDS and 50% formamide Filters were washed twice in 2 x NaCl/Cit/

0.1% SDS at room temperature for 15 min and then twice in

1 x NaCl/Cit/0.1%SDS and 0.5 x NaCl/Cit/0.1%SDS at

50 °C for 20 min

In situ hybridization analysis on foetal sections

*°§-Radiolabelled in situ hybridization was carried out as

follows: mouse embryos were sectioned at 8 uum and floated

onto TESPA (3-aminopropyltriethoxysilane) (Sigma)

coated slides The slides were pretreated with 5 pgemL"!

proteinase K (Sigma) and 2 mgmL"! glycine (Sigma) in

NaCl/P; and then re-fixed in 4% paraformaldehyde

(Sigma) Following treatment with 0.25% acetic anhydride

(BDH), hybridization was carried out overnight in a

humidified chamber at 55 °C The slides were then washed

at high stringency (20 min at 55 °C in 2x NaCl/Cit, 50%

formamide,10 mm dithiothreitol) and then treated with

40 ugmL7' RNAse A for 30 min at 37°C The high

stringency washes were repeated at 65 °C, followed by a

further wash in 0.1x NaCl/Cit/10 mm dithiothreitol, also at

65 °C The slides were then washed in 0.1x NaCl/Cit at

room temperature and dehydrated through 300 mm

ammonium acetate in 70% ethanol, 95% ethanol and then

100% ethanol Following air-drying, the slides were dipped

in Ilford K.5 photographic emulsion Autoradiography was

performed by exposing the sections in a light-proof box at

4 °C for 14 days The slides were then developed using

Kodak D19 developer, fixed with Kodak UNIFIX and

counter stained with malachite green Sections were photo-

graphed under dark field with an Olympus BH-2 micro-

scope and photographed with an Olympus camera using

Fujichrome 64T Tungsten film

Cloning of eukaryotic expression constructs

Full length CIB cDNA was PCR amplified from the yeast

GAL4 library pACT2 plasmid (Y198) and subcloned into

the pcDNA3.1 vector (Invitrogen) to produce the

C- terminal myc-tagged CIB construct CIB-myc The full-

length cDNA of human FEZ] was obtained as an IMAGE

clone from the UK HGMP Resource Centre and subcloned

into the pEGFP-N2 expression vector (Clontech) to produce

a C-terminal EGFP tagged construct (pFEZ1-EGFP) The

full-length NBR1I cDNA was subcloned into the pHM6

expression vector (Roche) to produce an N-terminal HA

epitope tagged construct (pHA-NBR1) All plasmids crea-

ted by PCR during the cloning steps were also sequenced

Immunoprecipitation and Western blotting

COS-7 cells were cultured in DMEM supplemented with

10% fetal calf serum (Life Technologies) Transient trans-

fection of eukaryotic expression vector constructs was

performed using the FuGENE™ reagent (Roche) Typi-

cally 1-2 ug of each construct was used to transfect COS-7

cells at 70% confluency in a six-well dish Sixteen hours

post-transfection, cells were washed once in ice cold NaCl/P;

and lysed at 4°C in 0.5 mL Tris/NaCl/P; lysis buffer

containing 137 mm NaCl, 20 mm Tris pH 8.0, 0.5% Tween

20, including a protease inhibitor cocktail (Complete™,

Roche) After lysis, cell homogenates were centrifuged at

16 000 g at 4 °C for 15 min and the supernatant collected

To confirm protein expression, 10 uL samples were sepa-

rated by SDS/PAGE and analysed by Western blotting Immunoprecipitations were carried out from 200 to 400 uL precleared cell lysate at 4 °C for 2 h with 2.5 ug of the anti- GFP Living Colors A.v.® peptide antibody (Clontech) and collected by protein LA agarose beads (Clontech) Beads were washed five times with lysis buffer and bound proteins were eluted by boiling Proteins were separated by SDS/ PAGE, transferred to an Immobilon-P membrane (Millipore) and immunoblotted with the appropriate antibody according to manufacturer’s instructions Western blots were developed by enhanced chemiluminescence (Amersham Pharmacia)

Cell transfection and immunofluorescence analysis COS-7 cells were transfected as described above and cultured for 16h before analysis for fluorescence as described previously [14] NBR1 was detected using either

HA mAb 12CAS (Roche) and TRITC or FITC conjugated goat anti-(mouse Ig) Ig (DAKO) or HA _ polyclonal antibody sc805 (Santa Cruz Biotechnology, Inc) followed

by TRITC or FITC conjugated swine anti-(rabbit Ig) Ig (DAKO) as appropriate CIB-myc was detected using the c-myc mAb 9E10 (Sigma) followed by TRITC or FITC conjugated goat anti-(mouse Ig) Ig (DAKO) Cell immu- nofluorescence was analysed using a LSM510 laser scanning confocal microscope (Zeiss)

RESULTS

NBR1 interacts with the PKC zeta interacting protein FEZ1 and the calcium and integrin binding protein CIB

In order to identify proteins that interact with NBRI, a yeast two-hybrid study was performed using full length human NBR1 as bait A human placental GAL4 AD fusion cDNA library was screened and a total of 2 x 10° clones were analysed Four positive clones were isolated, Y156, Y198, Y163 and Y214, which were positive by both the B-galactosidase assay and nutritional selection assays Sequence analysis of these four cDNA library clones showed that they represented two proteins, Y156, Y163 and Y214 all encoded partial cDNAs of FEZ1 and were all in frame with the GAL4 activation domain (GAL4 AD) The FEZ1 clones could be subdivided into clones encoding overlapping regions of amino acids 248-360 and 248-349 (Y214 and Y156, respectively), and the C-terminal amino acids 370-392 (Y163) Clone Y198 encoded the full-length cDNA of the calcium and integrin binding protein (CIB) in frame with the GAL4 AD

Mapping of regions involved in the interaction show that CIB and FEZ1 bind in the same domain

of NBR1

To identify which region of NBRI interacts with CIB or FEZ1, C-terminal deletions of NBRI were made and analysed in the GAL4 yeast two-hybrid system with Y 214, Y156, Y163 and Y198 The results of this analysis are shown in Fig 1B These constructs included the N-terminal

216 amino acids only (pGBTINBR1»16), the N-terminus,

ZZ and coiled-coil domain (pGBTINBR1333) and a C-terminal fragment encoding amino acids 494-849 which

contains none of these domains (pGBTONBR I¢term) AS

shown in Fig 1B, both CIB and FEZ1 interacted within the

C-terminus of NBR1 This region was then subdivided into

approximately 150 amino-acid fragments and further tested

by the yeast two-hybrid method (see Fig 1B, v—viii) Both

CIB and FEZ] interact strongly within the same region of

NBRI, encoding amino acids 451-597 and weakly with

amino acids 592-754 This region of NBR1 does not contain

any known or predicted functional domains The full

length NBRI coding region was also subcloned into the

GAL4-AD vector pGAD424 to produce the construct

pGAD424NBRI1 When this construct was tested in the

yeast two-hybrid assay, NBRI was also shown to form

homodimers We can conclude that this interaction occurs

between two domains, as protein products of both

PGBT9YNBRI1333 and pGBTONBRIcierm were able to

interact with full-length NBR1 (see Fig 1B, ijii,iv and vi)

FEZ1 and CIB interact with each other

To confirm the interactions between NBR1, CIB and FEZ1,

the full-length FEZ1 and CIB cDNAs were subcloned into

the GAL4-BD vector pGBKT7 (Clontech) and tested in the

yeast interaction assay with pGAD424NBRI The

pGBKT7-FEZ1 construct showed transcriptional activa-

tion function, and thus could not be used to verify the

NBR1/FEZ1 interaction, but the interaction between CIB

and NBRI was confirmed Using the yeast two-hybrid

assay, full length CIB and FEZ1 were also shown to bind

to each other This interaction was delineated to the

C-terminus of FEZ1 as Y163 (encoding amino acids

370-392) but not Y214 (encoding amino acids 248-360) of

FEZ] were shown to interact with full length CIB (data not

shown) FEZ1 was cloned as a mammalian homologue of

the C elegans UNC-76 protein involved in axonal out-

growth The two regions of FEZ1, which we have shown

can interact with NBRI, are part of domains that are

conserved between UNC-76 and rat FEZ1 [15]

Co-immunoprecipitation experiments confirm

that NBR1 interacts with FEZ1 /n vivo

Due to the lack of antibodies that could detect NBR1 and

either FEZ1 or CIB, we were unable to perform immuno-

precipitation or colocalization studies of endogenous

proteins Thus we exogenously expressed epitope-tagged

constructs, where COS-7 cells were either singly transfected

with pHA-NBRI, pFEZI-EGFP or cotransfected with

pHA-NBRI and either pFEZ1-EGFP or the empty vector

pEGFP-N2 that expresses EGFP only Immunoprecipitates

from cell lysates using a GFP polyclonal antibody were

separated by SDS/PAGE and Western blotted before

immunodetection using the HA mAb 12CA5 Figure 2D

shows that HA-NBRI is coimmunoprecipitated only in the

presence of FEZ1-EGFP (lanes 4) and not when expressed

with EGFP (lane 5) Panels A, B and C show Western

analysis of cell lysates to show comparable expression of

HA-NBRI1, FEZ1-EGFP and EGFP where expected These

results confirm that NBR1 and FEZ] interact in vivo The

interaction between NBRI and CIB was also tested by

immunoprecipitation with GST fusion proteins, in vitro

translated proteins and over-expressed proteins from trans-

fected COS-7 cells under several different buffer conditions,

EGFP - - - "`:

or

WB HÀ

Fig 2 Co-immunoprecipitation experiments confirm that NBRI and FEZ1 interact in vivo COS-7 cells (lane 1), COS-7 cells transfected with pHA-NBRI (dane 2), pFEZ1-EGFP (lane 3), pHA-NBRI and pFEZ1-EGFP (lane 4) and pHA-NBRI and pEGFP-N2 (lane 5) were assayed by Western blotting for expression of HA-NBRI (panel A), FEZ1-EGFP (panel B) and EGFP (panel C) Cell lysates were immunoprecipitated with the GFP polyclonal antibody and immuno- blotted with the HA antibody 12CAS5 (panel D)

but we were unable to confirm binding of CIB and NBR1

by other methods Nevertheless, given the data described in later sections of this paper, it is still likely that CIB and NBRI interact directly

Northern analysis of NBR1, CIB and FEZ1 expression

in human tissues

To address the question of whether the three proteins NBRI, CIB and FEZ] are expressed in the same tissues, Northern analysis was performed using human multiple tissue RNA blots (see Fig 3) Expression of NBR1 and CIB was shown to be widespread, whereas FEZ1, although expressed weakly in most of the tissues examined was most highly expressed in the brain As well as the more common 4.4-kb NBRI transcript observed in all tissues analysed, a smaller 4-kb RNA transcript of NBR1 was also present in testes This smaller transcript corresponds to the alternati- vely spliced Nbr1(la) transcript, which has been suggested

to be a testes-specific isoform [16] but was detected by RT- PCR in several other mouse tissues analysed (C Whitehouse, unpublished results) The ubiquitously expressed CIB transcript was1.2 kb in size and an additional transcript of approximately 1.5 kb was also observed in the testes which agrees with previous results [17] A 2.4-kb transcript of FEZ1 was also present in testes compared to the more common 2-kb mRNA, but the highest level of expression of FEZ1 was observed in the brain where a smaller transcript

of less than 1 kb was also present Thus NBR1 and CIB appear to be ubiquitously expressed in adult human tissues whereas FEZ1 expression is more restricted, although not exclusively, to the brain

Developmental pattern of expression of murine Nbr1 Using an antisense cDNA probe to murine Nbr1 encoding exons 16-3’ UTR, the developmental expression pattern of

tủ E < = z

860w >m3ế Bei ewe &

›£ œ 5œ 4Q *E200 8 oF

Bì m Ôn « S6 Ha “#242228 Zz

nm am ONO & m— he mt 6 6É

NBRI

FEZI

ACTIN

Fig 3 Northern blot analysis of expression patterns of CIB (panel A),

NBRI (panel B) and FEZ1 (panel C) in a panel of human adult tissues

Northern blots containing 2 pg per lane of poly(A)” RNA from

various adult human tissues were hybridized with cDNA probes

labelled with [a-*°P]dCTP The position of RNA size markers is indi-

cated on the left side of each blot Comparable loading of RNA in each

lane was shown by hybridization of a B-actin probe to the same blots

(panel d)

Nbr1 was analysed in mouse embryos by in situ hybridiza- tion (see Fig 4) The earliest sections analysed were at embryonic stage 9 d.p.c and show that Nbrl is widely expressed in all tissues However at embryonic stage 10.5— 13.5 d.p.c., the distribution of Nbrl RNA is shown to be largely restricted to the neural tube Northern analysis of Nbr expression in the adult mouse shows that expression is restored in all of the tissues analysed [18]

Development of the nervous system of the mouse embryo

is thought to begin at 7 d.p.c with the formation of the neural plate and is completed by 17 d.p.c The pattern of expression of Nbr1 in the developing embryo thus suggests that this gene may have a general role in early and late stages

of mouse development, but the restricted expression profile

at 10.5—13.5 d.p.c suggests a more specific role for Nbr1 in neural development This pattern of expression overlaps with the altered transcript profile of its interacting partner FEZ1, providing further evidence for the possible involve- ment of these two genes in a common cellular pathway, and more specifically in neuronal tissues

Subcellular colocalization of CIB, NBR1 and FEZ1 protein

The subcellular localization of NBR1, CIB and FEZ1 was analysed by transfection of COS-7 cells with epitope-tagged constructs pHA-NBR1, pcDNA3.1 CIB-myc and pFEZI- EGFP expression plasmids were singly or cotransfected into COS-7 cells and assayed 16 h later (see Fig 5) NBR1 was predominantly localized to the cytoplasm and was restricted

to a particulate, perinuclear fraction (Fig SA) FEZI- EGFP was also mainly localized to the cytoplasm, but

Fig 4 Jn situ hybridization analysis of Nbr1 expression during mouse embryonic develop- ment A Nbrl antisense probe covering exons

16-3’ UTR was labelled with [*°S]JdUTP and

incubated with embryonic mouse sections at (A) 9 d.p.c (B) 10.5 d.p.c (C) 11.5 đ.p.c and (D) 13.5 d.p.c N, neural tube; M, mandible;

B, brain; BV, brain ventricles

Fig 5 Intracellular localization of NBR1, CIB

and FEZ1 proteins in COS-7 cells Cells were

transfected either singly (a,b and c) or

cotransfected (dH) and processed for immu-

nofluorescence 16 h later (a) HA-NBRI (b)

FEZ1-EGFP (c) CIB-myc protein expression,

(df) coexpression of HA-NBRI1 and FEZ1-

EGFP, and (g-i) coexpression of HA-NBR1

and CIB-myc The pattern of CIB staining

observed in (g) was never observed in cells

expressing CIB-myc only Bar, 20 pm

showed a more diffuse pattern of expression than NBRI,

with some plasma membrane staining also present

(Fig 5B,D) Upon cotransfection of pFEZI-EGFP and

pHA-NBRI, both FEZ1-EGFP (Fig 5D) and HA-NBRI

(Fig SE) were shown to localize in the cytoplasm, and

showed very strong coexpression in the perinuclear com-

partment (yellow) (Fig 5F) Overexpression of HA-NBRI1

and FEZ1-EGFP in the same cell also often resulted in a

shift in HA-NBR1 expression to a more diffuse cytoplasmic

compartment similar to that observed for FEZ1-EGFP

alone (Fig SF)

CIB-myc showed a more complex pattern of expression,

with protein detected chiefly in the nucleus, but expression

was also observed in the cytoplasm or both nucleus and

cytoplasm (Fig 5C) This agrees with previous data that

shows CIB expression in both cellular compartments and is

consistent with the hypothesis that CIB is involved in

dynamic processes such as cell signalling pathways [19] In a

proportion of cells that coexpressed CIB and NBRI

however, the localization of CIB was drastically altered so

that it now completely colocalized with the perinuclear

pattern observed for NBR1 (Fig 51) This was not observed

however, in all cells that coexpressed CIB and NBR1, which

suggests that this may be a transient, dynamic association,

possibly linked to the stage of the cell cycle or signalling

status of the cell NBR1, when coexpressed with nuclear

CIB however, retained its perinuclear, cytoplasmic location

A similar shift in CIB expression pattern to the ER

compartment was observed with the presenilin protein PS2

[20] and to the cytoplasm when coexpressed with Snk [19]

Mutations in the presenilin genes PS/ and PS2 cause the

majority of cases of early onset Alzheimer’s disease [21]

Stimuli that induce synaptic plasticity result in increased expression of Fnk and Snk and lead to targeting of these proteins to the dendrites of activated neurons [19] The shift

in localization of CIB when coexpressed with NBR1 does however, provide persuasive evidence that NBRI and CIB

do interact in vivo

DISCUSSION Using a yeast two-hybrid approach, these experiments describe the identification of two proteins that interact with NBRI; CIB and FEZ1, which were also shown to interact with each other CIB was itself cloned by a yeast two-hybrid study using the integrin oy, subunit as bait [22] Sub- sequently, CIB has been shown to interact with a number of other proteins, including the polo-like kinases Fnk and Snk [19], DNA dependent kinase [17], and the Alzheimer’s disease presenilin 2 protein [20] CIB shows 58% amino-acid similarity with calcineurin B, the regulatory subunit of calcineurin (phosphatase 2B) and also shares 56% protein sequence similarity to calmodulin The protein contains two EF-hand motifs that bind Ca** and thus it has been suggested that the protein may act as a regulatory subunit of interacting partners [17] Northern analysis of CIB expres- sion showed that it is widely expressed in all tissues examined The subcellular localization of exogenously expressed CIB described herein agrees with previous studies, which have shown the accumulation in the nucleus and the cytoplasm of both transfected and endogenous CIB protein [19] The presence of CIB in different cellular compartments, and its ability to interact with proteins in the nucleus (DNA-PK), and cytoplasmic compartments (NBR1)

suggests that this protein may be involved in dynamic

processes or subcellular targeting of other proteins

Co-expression of NBR1 and CIB did not lead to targeting

of NBRI to the nucleus but did result in accumulation of

CIB in a proportion of cells to the perinuclear compartment

The functional significance of this change in localization is

now being studied

The interaction of NBRI1 with FEZ1 that was first

identified by the yeast two-hybrid assay was confirmed

in vivo by coimmunoprecipitation studies FEZ1 was also

identified by a yeast two-hybrid assay as a PKC zeta

interacting protein [15] PKC zeta is a member of the

atypical PKC family of serine/threonine protein kinases It

has been shown to be involved in a wide variety of

cellular processes, including signal transduction pathways

regulating cell proliferation, differentiation and apoptosis

FEZI1, by its interaction with PKC ¢ via its regulatory

domain, may be involved in influencing or determining

the subcellular localization or activity of this enzyme

Another protein, RBCK1, which as a member of the

RBCC family of proteins has structural similarities to

NBRI, was identified as a PKC beta I and PKC zeta-

interacting protein [23] This suggests the possibility that

similar members of the family of RBCC proteins may act

as downstream modifiers of PKC signalling pathways,

and the possibility that NBRI is involved in PKC zeta

signalling will be analysed

Several other proteins, including NBR1, the Drosophila

ref(2)p protein, rat PKC-zeta interacting protein (ZIP), a

novel interleukin-12 p40-related protein and a _ phos-

photyrosine-independent ligand of the pS5é6lck SH2

domain share a common domain composition and

organization, consisting of an octicosapeptide repeat

domain, a ZZ zinc finger and a_ ubiquitin-associated

domain, and thus have been suggested to be members of

a novel protein family [7] Further analysis of the

function of these proteins may demonstrate if there is a

common functional role for these family members in

signal transduction pathways

The changing pattern of expression of Nbrl during

murine development suggests that the protein may have a

more specific function during early development of the

murine neuronal tissues, and this is reflected in the

expression pattern of FEZ1 To further investigate the func-

tion of NBRI, a mouse knock-out model has been

produced, where the possible developmental effect of lack

of any Nbr! protein on neuronal development and function,

as well as susceptibility to cancer, is being monitored

ACKNOWLEDGEMENTS

We would like to acknowledge Chris Healy for interpretation of the

in situ results This work was supported by an MRC Programme Grant

No G6900577

REFERENCES

1 Campbell, I.G., Nicolai, H.M., Foulkes, W.D., Senger, G., Stamp,

G.W., Allan, G., Boyer, C., Jones, K., Bast, R.C Jr & Solomon,

E (1994) A novel gene encoding a B-box protein within the

BRCAI region at 17q21.1 Hum Mol Genet 3, 589-594

2 Bast, R.C Jr,, Klug, T.L., St John, E., Jenison, E., Niloff, J.M.,

Lazarus, H., Berkowitz, R.S., Leavitt, T., Griffiths, C.T., Parker,

L., Zurawski, V.R Jr & Knapp, R.C (1983) A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer New Engl J Med 309, 883-887

Yin, B.W & Lloyd, K.O (2001) Molecular cloning of the cal25 ovarian cancer antigen Identification as a new mucin, mucl6

J Biol Chem 276, 27371-27375

Nustad, K., Onsrud, M., Jansson, B & Warren, D (1998) CA

125 — epitopes and molecular size Int J Biol Markers 13, 196-199

Lenk, U., Oexle, K., Voit, T., Ancker, U., Hellner, K.A., Speer, A

& Hubner, C (1996) A cysteine 3340 substitution in the dystro- glycan-binding domain of dystrophin associated with Duchenne muscular dystrophy, mental retardation and absence of the ERG b-wave Hum Mol Genet 5, 973-975

Beck, K & Brodsky, B (1998) Supercoiled protein motifs: the collagen triple-helix and the alpha-helical coiled coil J Struct Biol 122, 17-29

Dimitrov, S.D., Matouskova, E & Forejt, J (2001) Expression of BRCA1I, NBR1 and NBR2 genes in human breast cancer cells Folia Biol 47, 120-127

Hofmann, K & Bucher, P (1996) The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway Trends Biochem Sci 21, 172-173

Ruffner, H., Joazeiro, C.A., Hemmati, D., Hunter, T & Verma, ILM (2001) Cancer-predisposing mutations within the RING domain of BRCAI: loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity Proc Natl Acad Sci USA 98, 5134-5139

Cao, T., Borden, K.L., Freemont, P.S & Etkin, L.D (1997) Involvement of the rfp tripartite motif in protein-protein interactions and subcellular distribution J Cell Sci 110, 1563-

1571

Ford, D., Easton, D.F., Stratton, M., Narod, S., Goldgar, D., Devilee, P., Bishop, D.T., Weber, B., Lenoir, G., Chang-Claude

et al (1998) Genetic heterogeneity and penetrance analysis of the BRCAI and BRCA2 genes in breast cancer families The Breast Cancer Linkage Consortium Am J Hum Genet 62, 676-689

Easton, D.F., Bishop, D.T., Ford, D & Crockford, G.P (1993) Genetic linkage analysis in familial breast and ovarian cancer: results from 214 families The Breast Cancer Linkage Consortium

Am J Hum Genet 52, 678-701

Breeden, L & Nasmyth, K (1985) Regulation of the yeast HO gene Cold Spring Harb Symp Quant Biol 50, 643-650 Whitehouse, C., Burchell, J., Gschmeissner, S., Brockhausen, I., Lloyd, K.O & Taylor-Papadimitriou, J (1997) A transfected sialyltransferase that is elevated in breast cancer and localizes to the medial/trans-Golgi apparatus inhibits the development of core-2-based O-glycans J Cell Biol 137, 1229-1241

Kuroda, S., Nakagawa, N., Tokunaga, C., Tatematsu, K & Tanizawa, K (1999) Mammalian homologue of the Caeno- rhabditis elegans UNC-76 protein involved in axonal outgrowth is

a protein kinase C zeta-interacting protein J Cell Biol 144, 403-411

Dimitrov, S., Brennerova, M & Forejt, J (2001) Expression profiles and intergenic structure of head-to-head oriented Brcal and Nbr] genes Gene 262, 89-98

Wu, X & Lieber, M.R (1997) Interaction between DNA-depen- dent protein kinase and a novel protein, KIP Mutat Res 385, 13-20

Chambers, J.A & Solomon, E (1996) Isolation of the murine NbrI gene adjacent to the murine Brcal gene Genomics 38, 305-313

Kauselmann, G., Weiler, M., Wulff, P., Jessberger, S., Konietzko, U., Scafidi, J., Staubli, U., Bereiter-Hahn, J., Strebhardt, K & Kuhl, D (1999) The polo-like protein kinases Fnk and Snk associate with a Ca(2+)- and integrin-binding protein and are

20

21

regulated dynamically with synaptic plasticity EMBO J 18,

5528-5539

Stabler, S.M., Ostrowski, L.L., Janicki, S.M & Monteiro, M.J

(1999) A myristoylated calctum-binding protein that preferentially

interacts with the Alzheimer’s disease presenilin 2 protein J Cell

Biol 145, 1277-1292

Cruts, M & Van Broeckhoven, C (1998) Presenilin mutations in

Alzheimer’s disease Hum Mutat 11, 183-190

22 Naik, U.P., Patel, P.M & Parise, L.V (1997) Identification of a

23

novel calcium-binding protein that interacts with the integrin alphalIb cytoplasmic domain J Biol Chem 272, 4651-4654 Tokunaga, C., Kuroda, S., Tatematsu, K., Nakagawa, N., Ono,

Y & Kikkawa, U (1998) Molecular cloning and characterization

of a novel protein kinase C- interacting protein with structural motifs related to RBCC family proteins Biochem Biophys Res Commun 244, 353-359.