BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 173, No. 1,1990
Pages 423-430
November 30,1990
P H O S P H O R Y L A T I O N OF THE H U M A N CELL P R O L I F E R A T I O N - A S S O C I A T E D N U C L E O L A R P R O T E I N PI20 B e n i g n o C. Valdez,
Rose K. Busch,
and Harris Busch
D e p a r t m e n t of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
Received October 18, 1990 The human cell p r o l i f e r a t i o n - a s s o c i a t e d nucleolar protein p120 was found in a variety of human cancer specimens but not in most normal resting cells. Polyclonal antibodies raised against b a c t e r i a l l y e x p r e s s e d p120 w e r e u s e d to i m m u n o p r e c i p i t a t e the p120 p r o t e i n i s o l a t e d from 3 2 p - l a b e l e d HeLa cells. The p120 p r o t e i n was p h o s p h o r y l a t e d at serine, t h r e o n i n e and t y r o s i n e residues. A tryptic peptide map showed it contained three labeled peptides. One of these p e p t i d e s c o m i g r a t e d w i t h a p120 peptide p h o s p h o r y l a t e d in v i t r o by casein kinase II. This peptide was p h o s p h o r y l a t e d in v i t r o b o t h at S e r - 1 8 1 and Thr-185. This region is juxtaposed to the epitope site recognized by the anti-pl20 monoclonal antibody. ©1990AcademicP .....Inc.
The p120 protein nucleolar
is a human cell p r o l i f e r a t i o n - a s s o c i a t e d
antigen with an apparent molecular weight of 120,000.
This antigen was detected not in most
normal
resting
in a variety cells
(i).
the early G1 phase of the cell cycle network of beaded microfibrils
of m a l i g n a n t
tumors
This protein
appears
in
(i, 2) and was observed in a
in the nucleolus
tance of p120 to cell proliferation
but
(3).
The impor-
was shown by the inhibition
of cell growth and DNA and RNA syntheses when anti-pl20 monoclonal antibody was m i c r o i n j e c t e d region recognized
into HeLa cells
by this anti-pl20
monoclonal
sequence EAAAGIQW located at amino acid residues Since p120 studied
is associated
its phosphorylation
ize its structure
and
(4).
The epitope
antibody has the 173-180
with cell proliferation,
as part of our efforts
function.
(5). we have
to character-
This was made possible
by the
Abbreviations: p120, protein with apparent m o l e c u l a r weight of 120,000; nt, nucleotide(s); bp, base pair(s); SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; NEM, Nethylmaleimide; MW, m o l e c u l a r weight; MBP, maltose binding protein; MBPI20.1 and MBPI20.21, maltose binding protein with p120 peptides.
423
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
p r o d u c t i o n of
anti-pl20 polyclonal
p120 expressed in E. coli.
antibodies
r a i s e d against
These antibodies were then used for
immunoprecipitation of in vivo phosphorylated p120.
One of the
three tryptic peptides of the in vivo phosphorylated p120 comigrated w i t h
a p120 p e p t i d e p h o s p h o r y l a t e d
in v i t r o
by c a s e i n
kinase II at Ser-181 and Thr-185. MATERIALS
AND
METHODS
Cloning and expression o_~fp120 cDNA. The full-length p120 cDNA was constructed from the longest partial p120 cDNA isolated from a fetal liver cDNA library (6) and from genomic fragments (7). The fetal liver p120 cDNA was digested with EcoRI, and the shorter fragment (cDNA/SF in Figure i) was isolated and partially digested with PstI. This fragment was subcloned into the PstI-
A
/
Xbal ~ ~00,
Pstl
~
~o== o
==
===8
U.I~
a.
~,LU
I
I I
EcoRI
i
I I
cDNA/SF
~Ligate
XbalNcol Pstl //~stl / ........~~
~ ~-
--
L
ii
cDNA/LF
_=
i
G3'
~Pstl (partial)
I--Pstl
I--EcoRI
Hindlll EcoRI B
III [---EcoRI
T7 Ncol ~EcoRI
.,n
l
~fLigate
Pstl EcoRI
I--Ncol t--EcoRI
~Ligate
I--Ncol ~EcoRI
Ncol Pstl / ~ ~ s t l
~Ligate Hindlll
~EcoR,
~--L__.~jst°p
2'//
J Ncol
Hindlll
=O°co..
~Ligate
\\
co.,
I._EcoRI
EcoRI
T ~ s t o
p
Ori",2-.~__/__~Apr (A) The first c o n s t r u c t i o n of full-length p120 eDNA. Figure i. half was c o n s t r u c t e d u s i n g the 5' g e n o m i c c l o n e (pUClBG5') and P s t l - p a r t i a l l y d i g e s t e d E c o R I f r a g m e n t of t h e c D N A ( c D N A / S F ) . (B) The second half was constructed using the 3' end of genomic c l o n e (G3') and E c o R I f r a g m e n t of the c D N A (insert in p T Z I 9 L F ) . p T Z 1 9 S H w a s d i g e s t e d w i t h EcoRI and the i n s e r t w a s s u b c l o n e d in pETFH.
424
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EcoRI s i t e s of p U C I 8 G 5 ' w h i c h c o n t a i n e d an X b a I - P s t I g e n o m i c f r a g m e n t (nt - 2 8 0 to 103), r e s u l t i n g in c l o n e p U C I 8 F H . The N c o I - E c o R I f r a g m e n t (nt 1 - 744 of p 1 2 0 cDNA) of p U C I 8 F H was s u b c l o n e d into p E T 8 c (8). The 3' half of the c D N A was cons t r u c t e d u s i n g two clones, p T Z I 9 L F w h i c h c o n t a i n e d the l o n g e r EcoRI f r a g m e n t of the fetal liver cDNA and pTZI9G3' w h i c h cont a i n e d the H i n d I I I - E c o R I fragment at the 3' end of the g e n o m i c clone (Figure IB). The r e s u l t i n g clone, pTZI9SH, c o n t a i n e d the p120 c o d i n g r e g i o n f r o m nt 745 t h r o u g h the s t o p c o d o n and an additional 400-bp 3' untranslated region. The insert in pTZI9SH was s u b c l o n e d at the E c o R I s i t e of p E T F H j o i n i n g the two c D N A fragments. The resulting clone, pET120, contained a full-length p120 cDNA sequence. T h e i n s e r t w a s e x p r e s s e d u s i n g E. c o l i strain BL26(DE3)pLysE according to Studier et al. (8). Production o_~f anti-pl20 polyclonal antibodies. Electrophoresis of E. coli extract containing expressed p120 protein was done on a preparative SDS-polyacrylamide (8%) gel and a region corres p o n d i n g to a m o l e c u l a r w e i g h t of 120,000 was excised. Protein p120 was e l e c t r o e l u t e d (9) from these gel slices, emulsified init i a l l y w i t h F r e u n d ' s c o m p l e t e adjuvant, and i n j e c t e d s u b c u t a n e ously into two rabbits. Incomplete adjuvant was used for subsequent i n j e c t i o n s w h i c h were given intradermally, s u b c u t a n e o u s l y and i n t r a m u s c u l a r l y . A p p r o x i m a t e l y 100-400 ~g of p r o t e i n were used per injection for a total of three injections per rabbit at 3-4 w e e k l y i n t e r v a l s . T h e a n i m a l s w e r e b o o s t e d a n d b l e d at monthly interval. A protein A affinity column (Pierce) was used to convert the rabbit serum to IgG. The sera were assayed by the E L I S A p r o c e d u r e (i0), W e s t e r n blot a n a l y s i s (Promega) and indirect immunofluorescence (i). R a d i o l a b e l i n g and i m m u n o p r e c i p i t a t i o n . HeLa cells in a 7080% c o n f l u e n t 150 ~ f l a s k was i n c u b a t e d in a p h o s p h a t e - or m e t h i o n i n e - f r e e m i n i m a l e s s e n t i a l m e d i u m w i t h 5% fetal b o v i n e serum (GIBCO) for 8 hours. The cells were then grown in a similar fresh m e d i u m containing either 0.8 - 1.0 mCi/ml [32p]-orthop h o s p h a t e (carrier-free) or 0.07 m C i / m l [ 3 5 S ] - m e t h i o n i n e (ICN) for 16-18 hrs. Nuclei were isolated as described p r e v i o u s l y (i) and lysed in triple-detergent lysis buffer according to Sambrook et al. (ii). To m i n i m i z e n o n s p e c i f i c immunocomplexes, 5 ~i p r e i m m u n e r a b b i t IgG and i00 ~i I m m u B i n d A b s o r b e n t (GENEX) were added to 500 ~i lysate, tumbled at 4°C for 1 hr, and centrifuged. To t h e p r e t r e a t e d lysate, i0 ~i r a b b i t i m m u n e IgG and i00 ~i ImmuBind Absorbent were added. The slurry was tumbled at 4°C for 1 hr and the immunocomplexes were separated, washed 4 times with RIPA buffer (Ii), and then with i0 mM Tris-cl (pH 7.5), 0.1% NP40, 1 mM PMSF, 2 mM NEM. P e l l e t s were r e s u s p e n d e d in 50 ~i 2X L a e m m l i b u f f e r c o n t a i n i n g 10% m e r c a p t o e t h a n o l , b o i l e d for 5 min and loaded onto SDS-polyacrylamide (8%) gel. C l o n i n g , e x p r e s s i o n and p u r i f i c a t i o n of m a l t o s e b i n d i n q p r o t e i n c o n t a i n i n q p120 peptides. F r a g m e n t s of p120 cDNA w e r e inserted at the BamHI site of pMalE178 (12). One fragment was a d o u b l e s t r a n d s y n t h e t i c o l i g o n u c l e o t i d e c o r r e s p o n d i n g to nt 505 to 555 of p120 cDNA with GATC 5' overhung in both ends. A second l o n g e r f r a g m e n t w a s p r o d u c e d by the p o l y m e r a s e c h a i n r e a c t i o n system. Amplification of nt 472 to 591 of p 1 2 0 c D N A was d o n e using synthetic oligonucleotide primers with BglII site. Maltose binding protein with or without p120 peptide was expressed in E_~. coli s t r a i n JMI09 as d e s c r i b e d (12) and p u r i f i e d in one step by affinity chromatography. B a c t e r i a l p e l l e t s w e r e s u s p e n d e d in Buffer A (i0 mM Tris-Cl (pH 7.2), 0.02% NAN3, 1 PMSF, 2 mM NEM), s o n i c a t e d and centrifuged. The clear lysate was l o a d e d onto a cross-linked amylose column (13) and washed with Buffer A. Bound 425
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein was eluted with i0 mM maltose in Buffer A and then concentrated using Centricon-30 (Amicon). In v i t r o labeling, p e p t i d e m a p p i n g and p h o s p h o a m i n o acid analysis. The casein kinase II was a gift of Drs. E. Cardellini and E. Durban, D e p a r t m e n t of Pharmacology, B a y l o r C o l l e g e of Medicine, Houston, TX, purified from HeLa nuclei (14). Kinase reactions were carried out in 60 ul of 50 mM Tris-Cl (pH 8.0), 50 mM NaCl, i0 mM MgCI2, 80 ~Ci of [T-32p]ATP LICN), 1-2 ~g of substrate and i0 ng of casein kinase II at 37 C for 30 min. Reaction was stopped by the addition of an equal volume of 2X Laemmli b u f f e r and then b o i l e d for 5 min. Peptide m a p p i n g and phosphoamino acid analysis were performed as described (15, 16).
RESULTS
The bacterially expressed protein from pET120 plasmid i~munoreacted with the anti-p120 monoclonal antibody and had the same migration on SDS-polyacrylamide gel as the p120 protein from HeLa cell nucleoli
(data not shown).
The patterns of partial diges-
tion using N-chlorosuccinimide/urea
for the two proteins were
essentially the same as shown by migration of peptides
on S D S - p o l y a c r y l a m i d e
gel
(6,
the immunoreactive
17).
The
antibodies raised against E. coli-expressed p120 acted w i t h the p120 results provide
isolated
from HeLa cell
polyclonal
also immunore-
nucleoli.
These
evidence that the p120 expressed from the cDNA
clone is essentially the same as the p120 protein isolated from HeLa cell nucleoli. The polyclonal antibodies against the bacterially expressed p120 protein immunoprecipited phosphorylated p120 protein.
Its
mobility on one-dimensional SDS-polyacrylamide gel was indistinguishable
from the immunoprecipitated
(Figure 2). IgG.
35S-labeled p120 protein
This protein was not precipitated by the preimmune
Both 32p_ and 35S-labeled precipitates were recognized by
the anti-pl20 monoclonal antibody.
These results show that p120
is a phosphoprotein recognized both by the monoclonal and polyclonal antibodies. The tryptic peptide map of p120 labeled in vivo contained three major phosphorylated peptides rived amino acid sequence of p120 residues
C-terminal
phosphorylated
The cDNA-de-
(6) showed clusters of acidic
to the s e r i n e
by c a s e i n kinase
(Figure 3A).
II
located between Ser-181 and Glu-191
or t h r e o n i n e (18,
19).
w h i c h m a y be
One c l u s t e r
(SEEETEDEEEE).
is
This region
is at the C-terminal of the epitope recognized by the anti-pl20 monoclonal antibody
(residues 173-180).
A DNA fragment coding
for the e p i t o p e
and this a c i d i c region was
maltose
p r o t e i n gene.
binding
426
inserted
The e x p r e s s e d
fusion
into the protein
V o l . 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
/
B
C
~
p120
Figure 2. Analysis of immunoprecipitates from 32p_ or 35S-labeled HeLa nuclear extracts. HeLa cells were labeled with [32p]-orthophosphate or [35S]-methionine and nuclear extracts were subjected to immunoprecipitation using anti-p120 polyclonal antibodies as described in Materials and Methods. Western blot analysis (A) of the immunoprecipitates was done using anti-p120 monoclonal antibody. SDS-polyacrylamide gel of 32p-labeled (B) and 35S-labeled (C) immunoprecipitates showed an antigen with an apparent molecular weight of 120,000 (p120).
(MBPI20.21 protein
in F i g u r e
(MBP)
MBPI20.21, kinase II vitro tide the
4) had the a c t i v i t y
for b i n d i n g to c r o s s - l i n k e d
but
not MBP,
was
phosphorylated
amylose.
phosphorylated
(lanes 1 and 3 in Figure 4C). MBPI20.21
The p u r i f i e d
i__nnv i t r o
by c a s e i n
The tryptic digest of in
contained
one
labeled p h o s p h o p e p -
(Figure 3B) which c o m i g r a t e d with one of the p e p t i d e s in vivo p h o s p h o r y l a t e d
3C).
of the m a l t o s e b i n d i n g
p120 tryptic digest
from
(spot 2 in Figure
This indicates the p o s s i b i l i t y that HeLa p120 is p h o s p h o r y -
lated by casein kinase II in at least one site. Either kinase II.
Ser-181
or T h r - 1 8 5
m a y be p h o s p h o r y l a t e d
by c a s e i n
The p h o s p h o a m i n o acid analysis of the i_nnvitro phosp-
h o r y l a t e d p e p t i d e showed that t h r e o n i n e was p h o s p h o r y l a t e d more
than
the
serine
vivo
phosphorylated
phosphothreonine
residue p120
(Figure
showed
it
and p h o s p h o t y r o s i n e A
B
5A).
Analysis
contained
4-fold
of the
phosphoserine,
(Figure 5B). C
Chr
D E1
Figure 3. Tryptic peptide maps of phosphorylated proteins. (A) In vivo phosphorylated p120 was immunoprecipitated from HeLa nuclear extract and treated with trypsin as described in Materials and Methods. Tryptic digests were analyzed by electrophoresis in the horizontal dimension (El) and by ascending chromatography (Chr). (B) Maltose binding protein containing p120 sequence (MBP120.21 in Figure 4) was phosphorylated in vitro using casein kinase II, digested with tyrpsin and analyzed as in (A). (C) Mixture of the tryptic digests of in vivo (A) and in vitro (B) phosphorylated proteins. 427
in
Vol. 173, No. 1, 1990
A
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
C
B !
2
3
4
!
2
3
I 2
3
4
N
A
B
P-Ser p120 Sequence Inserted in MBP
Clone MBP120.1
P-Thr
-QKAREAAAGIQW~EFE~ I
MBP120.21
~P-Tyr
-ALLPI ERAARKQKAREAAAGIQWSEEETEDEEEEKEVTPE-
Q
® Figure 4. Phosphorylation of maltose binding protein containing p120 sequences. The proteins were used as substrates for i__nn vitro phosphorylation by casein kinase II and analyzed by coomassie staining of SDS-polyacrylamide gel (A), Western blot using a n t i - p l 2 0 m o n o c l o n a l a n t i b o d y (B), and a u t o r a d i o g r a p h y (C). Lanes: i, maltose binding protein; 2, MBPI20.1; 3, MBPI20.21; 4, bovine serum albumin. (D) Sequence of p120 fragments inserted into a "permissive" site of the maltose binding protein as described in Materials and Methods. The epitope region recognized by the anti-pl20 monoclonal antibody is underlined. Arrows show the residues that may be p h o s p h o r y l a t e d by casein kinase If. Arrow heads show the tryptic digestion sites. Fiqure 5. Phosphoamino acid analysis. Tryptic digests of in vitro phosphorylated MBPI20.21 (A) or in vivo phosphorylated HeLa p120 (B) were hydrolyzed in 6 N HCI at 110°C for 2 hr under N 2 gas. The hydrolysate was mixed with phosphoamino acid standards and fractionated on cellulose sheets by high voltage eletrophoresis. Amino acids were stained with ninhydrin, and the 32p radioactivity was detected by autoradiography.
DISCUSSION The rapid
full-length
studies
on the
identification important Using
of
the bacterially
The present
which study
cDNA
constructed
structural
this
of c a n c e r
expressed
facilitated demonstrates
in t h i s
characterization
nucleolar
in t h e G1 p h a s e
generated
antigen
p120
antigen
p120,
that
is a p h o s p h o p r o t e i n .
428
and is
polyclonal
functional
cells
antibodies
characterization
this
permits
functionally
proliferating
and
further
which
study
of
(17). were p120.
proliferation-associated
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
To identify the possible site(s) casein kinase vitro.
Region
181-191 which
recognized by the anti-pl20 determine affect
monoclonal
the p120
site
protein
i_nn
region
antibody was examined to
and if this modification would
of the m o n o c l o n a l
of the m a l t o s e
retains
in p120,
peptide
to the epitope
antibody
This peptide was expressed by genetic
"permissive" hybrid
is C-terminal
if it was phosphorylated
the b i n d i n g
epitope.
of phosphorylation
II was used to phosphorylate
binding
the p r o p e r t i e s
to the
adjacent
insertion
protein
into a
(MBP).
The
of the w i l d t y p e M B P
so
that purification was possible in one step. The hybrid protein was phosphorylated with casein kinase II and digestion with trypsin yielded 3B).
This phosphopeptide
a p120 phosphopeptide
(Figure
comigrated with one of the three phos-
phopeptides
in the tryptic peptide map of in vivo phosphorylated
p120 w h i c h
indicates
vivo p h o s p h o r y l a t e d II. the
the p o s s i b l i t y at Thr-185
The phosphorylation binding
of
the
that H e L a
and/or
Ser-181
cell
p120
by casein
is in kinase
on Ser-181 and/or Thr-185 did not affect
anti-pl20
monoclonal
antibody
(data
not
shown). Casein kinase II has been found in high concentration nucleolus proteins
of mouse like B23,
tumor
cells
and
C23 and pp135
it p h o s p h o r y l a t e s
(20).
in the
nucleolar
Addition of a negatively
charged phosphate group in a negatively charged acidic domain may intensify the binding of these proteins to basic proteins. Figure
5B shows that the
also contained p h o s p h o t y r o s i n e phosphothreonine.
in vivo p h o s p h o r y l a t e d
HeLa p120
in addition to p h o s p h o s e r i n e
and
This implies that not only casein kinase II is
involved in the in vivo phosphorylation
of p120 but also a tyro-
sine kinase must phosphorylate p120 in vivo. ACKNOWLEDGMENTS
We thank Drs. E. Cardellini and E. Durban for casein kinase II, Dr. P. K. Chan for technical assistance, and Dr. F. Quiocho for the pMalE178 plasmid. This i n v e s t i g a t i o n was s u p p o r t e d by the C a n c e r R e s e a r c h Center Grant CA-I0893, awarded by the National Cancer Institute, D e p a r t m e n t of H e a l t h a n d H u m a n S e r v i c e s , U S P H S ; The D e B a k e y Medical Foundation; H. Leland Kaplan Cancer Research Endowment; Linda and Ronny Finger Cancer Research Endowment Fund; and The William S. Farish Fund.
REFERENCES
i.
Freeman, J. W., Busch, R. K., Gyorkey, F., Gyorkey, P., Ross, B.E., and Busch, H. (1988) Cancer Res. 48,1244-1251. 429
Vol. 173, No. 1, 1990
2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Jhiang, S., Yaneva, M., and Busch, H. (1990) Cell Growth Diff. 1,319-324. Ochs, R. L., Reilly, M.T., Freeman, J.W., and Busch, H. (1988) Cancer:Res. 48,6523-6529. Freeman, J. W., and Bondada, V. (1990) Proc. Am. Assoc. Cancer Res. 31,261. Valdez, B.C., Busch, R. K., Larson, R., and Busch, H. (1990) Cancer Res. 50,2704-2707. Fonagy, A., Henning, D., Jhiang, S., Haidar, M., Busch, R.K., Larson, R., Valdez, B.C, and Busch, H. (1989) Cancer Commun. 1,243-251. Larson, R. G., Henning, D., Haidar, M.A., Jhiang, S., Lin, W.L., Zhang, W.W., and Busch, H. (1990) C a n c e r Commun. 2,63-71. Studier, F. W., Rosenberg, A. H., and Dunn, J.J. (1990) Methods Enzymol. in press. Michalik, J., Yeoman, L. C., and Busch, H. (1981). Life sci. 20,1371-1379. Kelsey, D. E., Busch, R.K., and Busch, H. (1981) Cancer Lett. 12,295-303. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold spring Harbor, New York. Duplay, P., Szmelcman, S., Bedouelle, H., and Hofnung, M. (1987) J. Mol. Biol. 194,663-673. Ferenci, T., and Klotz, U. (1978) FEBS Letters. 94,213-217. Durban, E., Goodenough, M., Mills, J., and Busch, H. (1985) EMBO J. 4,2921-2926. Chan, P. K., Aldrich, M., Cook, R. G., and Busch, H. (1986) J. Biol. Chem. 261,1868-1872. Durban, E., Mills, J.S., Roll, D., and Busch, H. (1983) Biochem. Biophys. Res. Comm. 111,897-905. Busch, H. (1990) Cancer Res. 50,4830-4838. Edelman, A.M. (1987) Ann. Rev. Biochem. 56,567-613. Hathaway, G.M., and Traugh, J.A. (1982) Curr. Top. Cell. Reg. 21,101-127. Pfaff, M. and Anderer, F.A. (1988)/ Biochim. Biophys. Acta. 969,100-109.
430
Vol. 173, No. 1,1990
Pages 423-430
November 30,1990
P H O S P H O R Y L A T I O N OF THE H U M A N CELL P R O L I F E R A T I O N - A S S O C I A T E D N U C L E O L A R P R O T E I N PI20 B e n i g n o C. Valdez,
Rose K. Busch,
and Harris Busch
D e p a r t m e n t of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
Received October 18, 1990 The human cell p r o l i f e r a t i o n - a s s o c i a t e d nucleolar protein p120 was found in a variety of human cancer specimens but not in most normal resting cells. Polyclonal antibodies raised against b a c t e r i a l l y e x p r e s s e d p120 w e r e u s e d to i m m u n o p r e c i p i t a t e the p120 p r o t e i n i s o l a t e d from 3 2 p - l a b e l e d HeLa cells. The p120 p r o t e i n was p h o s p h o r y l a t e d at serine, t h r e o n i n e and t y r o s i n e residues. A tryptic peptide map showed it contained three labeled peptides. One of these p e p t i d e s c o m i g r a t e d w i t h a p120 peptide p h o s p h o r y l a t e d in v i t r o by casein kinase II. This peptide was p h o s p h o r y l a t e d in v i t r o b o t h at S e r - 1 8 1 and Thr-185. This region is juxtaposed to the epitope site recognized by the anti-pl20 monoclonal antibody. ©1990AcademicP .....Inc.
The p120 protein nucleolar
is a human cell p r o l i f e r a t i o n - a s s o c i a t e d
antigen with an apparent molecular weight of 120,000.
This antigen was detected not in most
normal
resting
in a variety cells
(i).
the early G1 phase of the cell cycle network of beaded microfibrils
of m a l i g n a n t
tumors
This protein
appears
in
(i, 2) and was observed in a
in the nucleolus
tance of p120 to cell proliferation
but
(3).
The impor-
was shown by the inhibition
of cell growth and DNA and RNA syntheses when anti-pl20 monoclonal antibody was m i c r o i n j e c t e d region recognized
into HeLa cells
by this anti-pl20
monoclonal
sequence EAAAGIQW located at amino acid residues Since p120 studied
is associated
its phosphorylation
ize its structure
and
(4).
The epitope
antibody has the 173-180
with cell proliferation,
as part of our efforts
function.
(5). we have
to character-
This was made possible
by the
Abbreviations: p120, protein with apparent m o l e c u l a r weight of 120,000; nt, nucleotide(s); bp, base pair(s); SDS, sodium dodecyl sulfate; PMSF, phenylmethylsulfonyl fluoride; NEM, Nethylmaleimide; MW, m o l e c u l a r weight; MBP, maltose binding protein; MBPI20.1 and MBPI20.21, maltose binding protein with p120 peptides.
423
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
p r o d u c t i o n of
anti-pl20 polyclonal
p120 expressed in E. coli.
antibodies
r a i s e d against
These antibodies were then used for
immunoprecipitation of in vivo phosphorylated p120.
One of the
three tryptic peptides of the in vivo phosphorylated p120 comigrated w i t h
a p120 p e p t i d e p h o s p h o r y l a t e d
in v i t r o
by c a s e i n
kinase II at Ser-181 and Thr-185. MATERIALS
AND
METHODS
Cloning and expression o_~fp120 cDNA. The full-length p120 cDNA was constructed from the longest partial p120 cDNA isolated from a fetal liver cDNA library (6) and from genomic fragments (7). The fetal liver p120 cDNA was digested with EcoRI, and the shorter fragment (cDNA/SF in Figure i) was isolated and partially digested with PstI. This fragment was subcloned into the PstI-
A
/
Xbal ~ ~00,
Pstl
~
~o== o
==
===8
U.I~
a.
~,LU
I
I I
EcoRI
i
I I
cDNA/SF
~Ligate
XbalNcol Pstl //~stl / ........~~
~ ~-
--
L
ii
cDNA/LF
_=
i
G3'
~Pstl (partial)
I--Pstl
I--EcoRI
Hindlll EcoRI B
III [---EcoRI
T7 Ncol ~EcoRI
.,n
l
~fLigate
Pstl EcoRI
I--Ncol t--EcoRI
~Ligate
I--Ncol ~EcoRI
Ncol Pstl / ~ ~ s t l
~Ligate Hindlll
~EcoR,
~--L__.~jst°p
2'//
J Ncol
Hindlll
=O°co..
~Ligate
\\
co.,
I._EcoRI
EcoRI
T ~ s t o
p
Ori",2-.~__/__~Apr (A) The first c o n s t r u c t i o n of full-length p120 eDNA. Figure i. half was c o n s t r u c t e d u s i n g the 5' g e n o m i c c l o n e (pUClBG5') and P s t l - p a r t i a l l y d i g e s t e d E c o R I f r a g m e n t of t h e c D N A ( c D N A / S F ) . (B) The second half was constructed using the 3' end of genomic c l o n e (G3') and E c o R I f r a g m e n t of the c D N A (insert in p T Z I 9 L F ) . p T Z 1 9 S H w a s d i g e s t e d w i t h EcoRI and the i n s e r t w a s s u b c l o n e d in pETFH.
424
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
EcoRI s i t e s of p U C I 8 G 5 ' w h i c h c o n t a i n e d an X b a I - P s t I g e n o m i c f r a g m e n t (nt - 2 8 0 to 103), r e s u l t i n g in c l o n e p U C I 8 F H . The N c o I - E c o R I f r a g m e n t (nt 1 - 744 of p 1 2 0 cDNA) of p U C I 8 F H was s u b c l o n e d into p E T 8 c (8). The 3' half of the c D N A was cons t r u c t e d u s i n g two clones, p T Z I 9 L F w h i c h c o n t a i n e d the l o n g e r EcoRI f r a g m e n t of the fetal liver cDNA and pTZI9G3' w h i c h cont a i n e d the H i n d I I I - E c o R I fragment at the 3' end of the g e n o m i c clone (Figure IB). The r e s u l t i n g clone, pTZI9SH, c o n t a i n e d the p120 c o d i n g r e g i o n f r o m nt 745 t h r o u g h the s t o p c o d o n and an additional 400-bp 3' untranslated region. The insert in pTZI9SH was s u b c l o n e d at the E c o R I s i t e of p E T F H j o i n i n g the two c D N A fragments. The resulting clone, pET120, contained a full-length p120 cDNA sequence. T h e i n s e r t w a s e x p r e s s e d u s i n g E. c o l i strain BL26(DE3)pLysE according to Studier et al. (8). Production o_~f anti-pl20 polyclonal antibodies. Electrophoresis of E. coli extract containing expressed p120 protein was done on a preparative SDS-polyacrylamide (8%) gel and a region corres p o n d i n g to a m o l e c u l a r w e i g h t of 120,000 was excised. Protein p120 was e l e c t r o e l u t e d (9) from these gel slices, emulsified init i a l l y w i t h F r e u n d ' s c o m p l e t e adjuvant, and i n j e c t e d s u b c u t a n e ously into two rabbits. Incomplete adjuvant was used for subsequent i n j e c t i o n s w h i c h were given intradermally, s u b c u t a n e o u s l y and i n t r a m u s c u l a r l y . A p p r o x i m a t e l y 100-400 ~g of p r o t e i n were used per injection for a total of three injections per rabbit at 3-4 w e e k l y i n t e r v a l s . T h e a n i m a l s w e r e b o o s t e d a n d b l e d at monthly interval. A protein A affinity column (Pierce) was used to convert the rabbit serum to IgG. The sera were assayed by the E L I S A p r o c e d u r e (i0), W e s t e r n blot a n a l y s i s (Promega) and indirect immunofluorescence (i). R a d i o l a b e l i n g and i m m u n o p r e c i p i t a t i o n . HeLa cells in a 7080% c o n f l u e n t 150 ~ f l a s k was i n c u b a t e d in a p h o s p h a t e - or m e t h i o n i n e - f r e e m i n i m a l e s s e n t i a l m e d i u m w i t h 5% fetal b o v i n e serum (GIBCO) for 8 hours. The cells were then grown in a similar fresh m e d i u m containing either 0.8 - 1.0 mCi/ml [32p]-orthop h o s p h a t e (carrier-free) or 0.07 m C i / m l [ 3 5 S ] - m e t h i o n i n e (ICN) for 16-18 hrs. Nuclei were isolated as described p r e v i o u s l y (i) and lysed in triple-detergent lysis buffer according to Sambrook et al. (ii). To m i n i m i z e n o n s p e c i f i c immunocomplexes, 5 ~i p r e i m m u n e r a b b i t IgG and i00 ~i I m m u B i n d A b s o r b e n t (GENEX) were added to 500 ~i lysate, tumbled at 4°C for 1 hr, and centrifuged. To t h e p r e t r e a t e d lysate, i0 ~i r a b b i t i m m u n e IgG and i00 ~i ImmuBind Absorbent were added. The slurry was tumbled at 4°C for 1 hr and the immunocomplexes were separated, washed 4 times with RIPA buffer (Ii), and then with i0 mM Tris-cl (pH 7.5), 0.1% NP40, 1 mM PMSF, 2 mM NEM. P e l l e t s were r e s u s p e n d e d in 50 ~i 2X L a e m m l i b u f f e r c o n t a i n i n g 10% m e r c a p t o e t h a n o l , b o i l e d for 5 min and loaded onto SDS-polyacrylamide (8%) gel. C l o n i n g , e x p r e s s i o n and p u r i f i c a t i o n of m a l t o s e b i n d i n q p r o t e i n c o n t a i n i n q p120 peptides. F r a g m e n t s of p120 cDNA w e r e inserted at the BamHI site of pMalE178 (12). One fragment was a d o u b l e s t r a n d s y n t h e t i c o l i g o n u c l e o t i d e c o r r e s p o n d i n g to nt 505 to 555 of p120 cDNA with GATC 5' overhung in both ends. A second l o n g e r f r a g m e n t w a s p r o d u c e d by the p o l y m e r a s e c h a i n r e a c t i o n system. Amplification of nt 472 to 591 of p 1 2 0 c D N A was d o n e using synthetic oligonucleotide primers with BglII site. Maltose binding protein with or without p120 peptide was expressed in E_~. coli s t r a i n JMI09 as d e s c r i b e d (12) and p u r i f i e d in one step by affinity chromatography. B a c t e r i a l p e l l e t s w e r e s u s p e n d e d in Buffer A (i0 mM Tris-Cl (pH 7.2), 0.02% NAN3, 1 PMSF, 2 mM NEM), s o n i c a t e d and centrifuged. The clear lysate was l o a d e d onto a cross-linked amylose column (13) and washed with Buffer A. Bound 425
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
protein was eluted with i0 mM maltose in Buffer A and then concentrated using Centricon-30 (Amicon). In v i t r o labeling, p e p t i d e m a p p i n g and p h o s p h o a m i n o acid analysis. The casein kinase II was a gift of Drs. E. Cardellini and E. Durban, D e p a r t m e n t of Pharmacology, B a y l o r C o l l e g e of Medicine, Houston, TX, purified from HeLa nuclei (14). Kinase reactions were carried out in 60 ul of 50 mM Tris-Cl (pH 8.0), 50 mM NaCl, i0 mM MgCI2, 80 ~Ci of [T-32p]ATP LICN), 1-2 ~g of substrate and i0 ng of casein kinase II at 37 C for 30 min. Reaction was stopped by the addition of an equal volume of 2X Laemmli b u f f e r and then b o i l e d for 5 min. Peptide m a p p i n g and phosphoamino acid analysis were performed as described (15, 16).
RESULTS
The bacterially expressed protein from pET120 plasmid i~munoreacted with the anti-p120 monoclonal antibody and had the same migration on SDS-polyacrylamide gel as the p120 protein from HeLa cell nucleoli
(data not shown).
The patterns of partial diges-
tion using N-chlorosuccinimide/urea
for the two proteins were
essentially the same as shown by migration of peptides
on S D S - p o l y a c r y l a m i d e
gel
(6,
the immunoreactive
17).
The
antibodies raised against E. coli-expressed p120 acted w i t h the p120 results provide
isolated
from HeLa cell
polyclonal
also immunore-
nucleoli.
These
evidence that the p120 expressed from the cDNA
clone is essentially the same as the p120 protein isolated from HeLa cell nucleoli. The polyclonal antibodies against the bacterially expressed p120 protein immunoprecipited phosphorylated p120 protein.
Its
mobility on one-dimensional SDS-polyacrylamide gel was indistinguishable
from the immunoprecipitated
(Figure 2). IgG.
35S-labeled p120 protein
This protein was not precipitated by the preimmune
Both 32p_ and 35S-labeled precipitates were recognized by
the anti-pl20 monoclonal antibody.
These results show that p120
is a phosphoprotein recognized both by the monoclonal and polyclonal antibodies. The tryptic peptide map of p120 labeled in vivo contained three major phosphorylated peptides rived amino acid sequence of p120 residues
C-terminal
phosphorylated
The cDNA-de-
(6) showed clusters of acidic
to the s e r i n e
by c a s e i n kinase
(Figure 3A).
II
located between Ser-181 and Glu-191
or t h r e o n i n e (18,
19).
w h i c h m a y be
One c l u s t e r
(SEEETEDEEEE).
is
This region
is at the C-terminal of the epitope recognized by the anti-pl20 monoclonal antibody
(residues 173-180).
A DNA fragment coding
for the e p i t o p e
and this a c i d i c region was
maltose
p r o t e i n gene.
binding
426
inserted
The e x p r e s s e d
fusion
into the protein
V o l . 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
/
B
C
~
p120
Figure 2. Analysis of immunoprecipitates from 32p_ or 35S-labeled HeLa nuclear extracts. HeLa cells were labeled with [32p]-orthophosphate or [35S]-methionine and nuclear extracts were subjected to immunoprecipitation using anti-p120 polyclonal antibodies as described in Materials and Methods. Western blot analysis (A) of the immunoprecipitates was done using anti-p120 monoclonal antibody. SDS-polyacrylamide gel of 32p-labeled (B) and 35S-labeled (C) immunoprecipitates showed an antigen with an apparent molecular weight of 120,000 (p120).
(MBPI20.21 protein
in F i g u r e
(MBP)
MBPI20.21, kinase II vitro tide the
4) had the a c t i v i t y
for b i n d i n g to c r o s s - l i n k e d
but
not MBP,
was
phosphorylated
amylose.
phosphorylated
(lanes 1 and 3 in Figure 4C). MBPI20.21
The p u r i f i e d
i__nnv i t r o
by c a s e i n
The tryptic digest of in
contained
one
labeled p h o s p h o p e p -
(Figure 3B) which c o m i g r a t e d with one of the p e p t i d e s in vivo p h o s p h o r y l a t e d
3C).
of the m a l t o s e b i n d i n g
p120 tryptic digest
from
(spot 2 in Figure
This indicates the p o s s i b i l i t y that HeLa p120 is p h o s p h o r y -
lated by casein kinase II in at least one site. Either kinase II.
Ser-181
or T h r - 1 8 5
m a y be p h o s p h o r y l a t e d
by c a s e i n
The p h o s p h o a m i n o acid analysis of the i_nnvitro phosp-
h o r y l a t e d p e p t i d e showed that t h r e o n i n e was p h o s p h o r y l a t e d more
than
the
serine
vivo
phosphorylated
phosphothreonine
residue p120
(Figure
showed
it
and p h o s p h o t y r o s i n e A
B
5A).
Analysis
contained
4-fold
of the
phosphoserine,
(Figure 5B). C
Chr
D E1
Figure 3. Tryptic peptide maps of phosphorylated proteins. (A) In vivo phosphorylated p120 was immunoprecipitated from HeLa nuclear extract and treated with trypsin as described in Materials and Methods. Tryptic digests were analyzed by electrophoresis in the horizontal dimension (El) and by ascending chromatography (Chr). (B) Maltose binding protein containing p120 sequence (MBP120.21 in Figure 4) was phosphorylated in vitro using casein kinase II, digested with tyrpsin and analyzed as in (A). (C) Mixture of the tryptic digests of in vivo (A) and in vitro (B) phosphorylated proteins. 427
in
Vol. 173, No. 1, 1990
A
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
C
B !
2
3
4
!
2
3
I 2
3
4
N
A
B
P-Ser p120 Sequence Inserted in MBP
Clone MBP120.1
P-Thr
-QKAREAAAGIQW~EFE~ I
MBP120.21
~P-Tyr
-ALLPI ERAARKQKAREAAAGIQWSEEETEDEEEEKEVTPE-
Q
® Figure 4. Phosphorylation of maltose binding protein containing p120 sequences. The proteins were used as substrates for i__nn vitro phosphorylation by casein kinase II and analyzed by coomassie staining of SDS-polyacrylamide gel (A), Western blot using a n t i - p l 2 0 m o n o c l o n a l a n t i b o d y (B), and a u t o r a d i o g r a p h y (C). Lanes: i, maltose binding protein; 2, MBPI20.1; 3, MBPI20.21; 4, bovine serum albumin. (D) Sequence of p120 fragments inserted into a "permissive" site of the maltose binding protein as described in Materials and Methods. The epitope region recognized by the anti-pl20 monoclonal antibody is underlined. Arrows show the residues that may be p h o s p h o r y l a t e d by casein kinase If. Arrow heads show the tryptic digestion sites. Fiqure 5. Phosphoamino acid analysis. Tryptic digests of in vitro phosphorylated MBPI20.21 (A) or in vivo phosphorylated HeLa p120 (B) were hydrolyzed in 6 N HCI at 110°C for 2 hr under N 2 gas. The hydrolysate was mixed with phosphoamino acid standards and fractionated on cellulose sheets by high voltage eletrophoresis. Amino acids were stained with ninhydrin, and the 32p radioactivity was detected by autoradiography.
DISCUSSION The rapid
full-length
studies
on the
identification important Using
of
the bacterially
The present
which study
cDNA
constructed
structural
this
of c a n c e r
expressed
facilitated demonstrates
in t h i s
characterization
nucleolar
in t h e G1 p h a s e
generated
antigen
p120
antigen
p120,
that
is a p h o s p h o p r o t e i n .
428
and is
polyclonal
functional
cells
antibodies
characterization
this
permits
functionally
proliferating
and
further
which
study
of
(17). were p120.
proliferation-associated
Vol. 173, No. 1, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
To identify the possible site(s) casein kinase vitro.
Region
181-191 which
recognized by the anti-pl20 determine affect
monoclonal
the p120
site
protein
i_nn
region
antibody was examined to
and if this modification would
of the m o n o c l o n a l
of the m a l t o s e
retains
in p120,
peptide
to the epitope
antibody
This peptide was expressed by genetic
"permissive" hybrid
is C-terminal
if it was phosphorylated
the b i n d i n g
epitope.
of phosphorylation
II was used to phosphorylate
binding
the p r o p e r t i e s
to the
adjacent
insertion
protein
into a
(MBP).
The
of the w i l d t y p e M B P
so
that purification was possible in one step. The hybrid protein was phosphorylated with casein kinase II and digestion with trypsin yielded 3B).
This phosphopeptide
a p120 phosphopeptide
(Figure
comigrated with one of the three phos-
phopeptides
in the tryptic peptide map of in vivo phosphorylated
p120 w h i c h
indicates
vivo p h o s p h o r y l a t e d II. the
the p o s s i b l i t y at Thr-185
The phosphorylation binding
of
the
that H e L a
and/or
Ser-181
cell
p120
by casein
is in kinase
on Ser-181 and/or Thr-185 did not affect
anti-pl20
monoclonal
antibody
(data
not
shown). Casein kinase II has been found in high concentration nucleolus proteins
of mouse like B23,
tumor
cells
and
C23 and pp135
it p h o s p h o r y l a t e s
(20).
in the
nucleolar
Addition of a negatively
charged phosphate group in a negatively charged acidic domain may intensify the binding of these proteins to basic proteins. Figure
5B shows that the
also contained p h o s p h o t y r o s i n e phosphothreonine.
in vivo p h o s p h o r y l a t e d
HeLa p120
in addition to p h o s p h o s e r i n e
and
This implies that not only casein kinase II is
involved in the in vivo phosphorylation
of p120 but also a tyro-
sine kinase must phosphorylate p120 in vivo. ACKNOWLEDGMENTS
We thank Drs. E. Cardellini and E. Durban for casein kinase II, Dr. P. K. Chan for technical assistance, and Dr. F. Quiocho for the pMalE178 plasmid. This i n v e s t i g a t i o n was s u p p o r t e d by the C a n c e r R e s e a r c h Center Grant CA-I0893, awarded by the National Cancer Institute, D e p a r t m e n t of H e a l t h a n d H u m a n S e r v i c e s , U S P H S ; The D e B a k e y Medical Foundation; H. Leland Kaplan Cancer Research Endowment; Linda and Ronny Finger Cancer Research Endowment Fund; and The William S. Farish Fund.
REFERENCES
i.
Freeman, J. W., Busch, R. K., Gyorkey, F., Gyorkey, P., Ross, B.E., and Busch, H. (1988) Cancer Res. 48,1244-1251. 429
Vol. 173, No. 1, 1990
2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Jhiang, S., Yaneva, M., and Busch, H. (1990) Cell Growth Diff. 1,319-324. Ochs, R. L., Reilly, M.T., Freeman, J.W., and Busch, H. (1988) Cancer:Res. 48,6523-6529. Freeman, J. W., and Bondada, V. (1990) Proc. Am. Assoc. Cancer Res. 31,261. Valdez, B.C., Busch, R. K., Larson, R., and Busch, H. (1990) Cancer Res. 50,2704-2707. Fonagy, A., Henning, D., Jhiang, S., Haidar, M., Busch, R.K., Larson, R., Valdez, B.C, and Busch, H. (1989) Cancer Commun. 1,243-251. Larson, R. G., Henning, D., Haidar, M.A., Jhiang, S., Lin, W.L., Zhang, W.W., and Busch, H. (1990) C a n c e r Commun. 2,63-71. Studier, F. W., Rosenberg, A. H., and Dunn, J.J. (1990) Methods Enzymol. in press. Michalik, J., Yeoman, L. C., and Busch, H. (1981). Life sci. 20,1371-1379. Kelsey, D. E., Busch, R.K., and Busch, H. (1981) Cancer Lett. 12,295-303. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold spring Harbor, New York. Duplay, P., Szmelcman, S., Bedouelle, H., and Hofnung, M. (1987) J. Mol. Biol. 194,663-673. Ferenci, T., and Klotz, U. (1978) FEBS Letters. 94,213-217. Durban, E., Goodenough, M., Mills, J., and Busch, H. (1985) EMBO J. 4,2921-2926. Chan, P. K., Aldrich, M., Cook, R. G., and Busch, H. (1986) J. Biol. Chem. 261,1868-1872. Durban, E., Mills, J.S., Roll, D., and Busch, H. (1983) Biochem. Biophys. Res. Comm. 111,897-905. Busch, H. (1990) Cancer Res. 50,4830-4838. Edelman, A.M. (1987) Ann. Rev. Biochem. 56,567-613. Hathaway, G.M., and Traugh, J.A. (1982) Curr. Top. Cell. Reg. 21,101-127. Pfaff, M. and Anderer, F.A. (1988)/ Biochim. Biophys. Acta. 969,100-109.
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