 |
INTRODUCTION |
The Jak1-Stat pathway
plays a crucial role in the signal transduction of many cytokines,
growth factors and hormones. Central to this pathway are the Jak family
of protein tyrosine kinases. This family includes the mammalian kinases
Jak1, Jak2, Jak3, and Tyk2 and the Drosophila melanogaster
kinase encoded by the hopscotch (hop) locus
(1-14). The Jaks are essential for the biological activities mediated
by these ligands and defects in this family of kinases have been shown
to lead to a number of disease states in both mammals and D. melanogaster (15-23).
The role of the Jak kinases in cytokine signal transduction was first
shown for the interferons (IFNs) (24, 25). Subsequently, many reports
have demonstrated that Jak activation occurs rapidly after ligand
stimulation (1, 8, 9, 26). This activation initiates a cascade of
events, which includes receptor phosphorylation and recruitment,
subsequent phosphorylation and nuclear translocation of members of the
Stat (signal transducers and activators of transcription) family of
proteins, which then activate cytokine-inducible genes (27). In
addition to their enzymatic role, several reports have demonstrated
that the Jaks play a structural role in the receptor complex and that
the Jaks may have functions in addition to their kinase activity that
are important for signaling. For example, introduction of a
kinase-inactive mutant of Jak1 into cells that lack this kinase (and
are unresponsive to interferon-
(IFN-) restores partial
IFN--induced gene expression (28, 29). Furthermore, the amino
terminus of Tyk2 stabilizes the IFNAR1 chain of the IFN-
receptor
complex (30).
In addition to their interactions with cytokine receptor chains, a
large body of evidence has accumulated demonstrating that the Jak
kinases interact with other signaling proteins. In particular, Jak2 was
reported to interact with SHPTP1, SHPTP2, PP2A, PI3K, Yes, Fyn, Shc,
Syp, Grb2, the angiotensin II AT1 receptor, and the serotonin 5-HT2A
receptor (31-44). The ability to interact with such diverse proteins
underscores the complex role of Jak2, which is activated by the
majority of ligands that utilize the Jak-Stat pathway (45). While the
physiological roles for these interactions have not been characterized,
they suggest that the Jaks play a role in other pathways and/or
facilitate cross-talk between signaling pathways.
In identifying Jak2-interacting proteins with the yeast two-hybrid
system, we cloned a human homologue of the Schizosaccaromyces pombe Skb1 protein and the Saccharomyces cerevisiae
protein encoded by the HSL7 gene (46-48). The
skb1 gene was initially identified during a two-hybrid
screen for proteins interacting with the shk1 kinase which represents a
member of the
p21cdc42/Rac1-activated
kinase (PAK) family of protein kinases (47). Recent data
suggest that removal of this protein results in cell cycle abnormalities and that the human homologue of this protein can functionally substitute for Skb1
(49).2 The HSL7
(histone synthetic lethal 7) gene was initially identified as a gene
whose mutation is lethal in combination with a mutation in the histone
H3 and was described to be a negative regulator of Swe1 function (48).
Disruption of HSL7 also results in cell cycle abnormalities
of S. cerevisiae (48). Taken together these data suggest
that this family of proteins is involved in coordinating cellular
events such as the cell cycle or cellular signaling. Since no
functional motifs or biochemical activities had been identified for
Skb1 or Hsl7p, we focused on identifying a biochemical activity for
JBP1. This report shows that JBP1 is a protein methyltransferase.
| |
MATERIALS AND METHODS |
Creation of Plasmid Encoding
GAL4DBD-Jak2--
Expression vectors for all the Jak
kinases (Jak1, Jak2, Jak3,and Tyk2) were gifts from James Ihle and John
Krowlewski. The two-hybrid system vector pAS2 which contains the yeast
tryptophan (TRP1)-selectable marker and a hemagglutinin (HA) tag, was a
gift from Stephan Elledge (50). To create pAS2-Jak2, the murine Jak2 cDNA was modified in the following manner. First, the
BsrGI site in the 3'-untranslated region of the plasmid
BluescriptSK-muJak2 (a gift from James Ihle) was removed by digesting
with NheI, blunt ending with the large fragment of DNA
polymerase I (Klenow fragment), digesting with EcoRV,and
recircularizing with T4 DNA ligase. This created the plasmid
muJak2-BsrGI. A linker containing a SfiI site was
placed into the remaining BsrGI site located 53 base pairs downstream from the translational start codon. This linker was created
by annealing two oligonucleotides (5'-GTACGGCCATGGAGGCC-3' and
5'-GTACGGCCTCCATGGCC-3'), then heating equimolar amounts to 100 °C,
and cooling slowly to 4 °C in 10 mM Tris·Cl (pH 7.8), 10 mM MgCl2. The annealed linker was ligated
into the BsrGI-digested muJak2-BsrGI plasmid.
This created the plasmid muJak2-SfiI. The Jak2 cDNA was
cloned from muJak2-SfiI into pAS2 as a
SfiI-SalI fragment. This created the plasmid
pAS2-Jak2, which encoded the GAL4 DNA binding domain fused to amino
acids 19-1129 of murine Jak2.
Yeast Two-hybrid System and Cloning of JBP1--
All two-hybrid
system materials except the two-hybrid system HeLa cell cDNA
library were gifts from Stephan Elledge (50). The HeLa library in
vector pGADH, which contains the LEU2 selectable marker, was a gift
from Greg Hannon (51). The GAL4 activation domain was fused to the HeLa
cell cDNAs in this library. Yeast strains used were Y190
(MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52
leu2-3,-112 +URA3:: GAL
lacZ,
LYS2::GAL(UAS)HIS3 cyhr)
and Y187 (MAT gal4 gal80 his3 trp1-901
ade2-101 ura3-52 leu2-3,-112 met-
URA3::GALlacZ). Yeast
transformations were performed via the lithium acetate method as
described previously (52). The transformed yeast cells were plated onto
agar media lacking leucine, tryptophan, and histidine and containing 30 mM 3-aminotriazole to isolate colonies containing both
plasmids and activating the HIS3 reporter gene. After 7 days at
30 °C, 143 histidine prototrophs were selected and restreaked onto
media lacking leucine and tryptophan. These were assayed for
-galactosidase expression with the paper filter assay (52). Colonies
that showed evidence of -galactosidase expression were streaked onto
media lacking leucine and containing 10 µg/ml cycloheximide to select
for loss of the pAS2-Jak2 plasmid. Colonies from these plates were
restreaked onto media lacking leucine and tryptophan to confirm loss of
the pAS2-Jak2 plasmid. One to three colonies of each isolate containing
the library plasmid without the pAS2-Jak2 plasmid were each mated to
yeast strain Y187 containing the plasmid pAS1-CDK2, pAS1-SNF1,
pAS1-lamin, or pAS2-Jak2. Diploids from these matings were assayed for
-galactosidase expression by the paper filter method. Plasmid DNA
from the clones showing evidence of -galactosidase expression only
in the presence of Jak2 were rescued, characterized by restriction
endonuclease digestion, and sequenced. The two plasmids corresponding
to JBP1 were initially called p31-2B and p41-3A and had inserts of
approximately 1500 base pairs (46).
To isolate full-length clones corresponding to the cDNA fragments
within plasmids p31-2B and p41-3A, the insert from plasmid p41-3A
was released with the EcoRI and XhoI restriction
endonucleases. This fragment was labeled by the random hexamer method
and used to identify hybridizing sequences in a human 
cDNA
library (53, 54). Positive phage plaques were purified and plasmid DNA
rescued by digesting total DNA with either NotI or
MluI restriction endonucleases, recircularizing with T4 DNA
ligase and transforming into Escherichia coli. Inserts were
sequenced with vector oligonucleotides, internal oligonucleotides, and
fragment cloning into pBluescript SK
(Stratagene). The largest
cDNA isolated was in phagemid p41-6. The insert from phagemid
p41-6 was cloned as a SalI fragment into the compatible
XhoI site of pBluescript SK to yield plasmid
p41-6-Bluescript.
Construction of Expression Vectors--
The plasmid
pcDNA3-HA was created by amplifying a triple influenza HA tag from
vector pSM491 which was a gift from Terry Goss Kinzy (55). This was
accomplished by polymerase chain reaction (PCR) with the following
oligonucleotides: HA-5' (5'-GCCGGTACCATGGGCCGCATCTTTTACCCA-3') and HA-AS (5'ACCACGGAGCCTTTGCTGGAGCGGCCGCACTGAGCAGCGT-3'). Thirty cycles of PCR were used under the following conditions: 20 µM of each oligonucleotide, 0.025 µg of template
plasmid DNA, 0.5 units of Taq DNA polymerase, with buffer
containing 10 mM Tris·HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3 at 20 °C) in a total volume
of 0.05 ml. Thirty cycles at 94 °C for 30 s, 50 °C for
45 s, and 72 °C for 45 s were performed. The product from
this reaction was digested sequentially with NotI and
KpnI restriction endonucleases and cloned into vector
pcDNA3 (Invitrogen), which was digested with NotI and
KpnI restriction endonucleases. To create plasmid pEF2-HA,
plasmid pcDNA3-HA was digested with KpnI and
SfuI restriction endonucleases and the fragment containing
the HA tag and multiple cloning site was cloned into vector pcDEF3
digested with the same enzymes (56).
The plasmid to express HA-tagged JBP1 was constructed in three steps.
First, PCR was used to amplify the insert with a 5' NotI
site whose frame was compatible with the NotI site of vector pcDNA3-HA. This was accomplished by PCR with the plasmid
p41-6-Bluescript as the template and the following 3' and 5'
oligonucleotide primers, respectively: U2-5-4
(5'-TTGTGCCACCACATCCACGT-3') and HA-41-5' (5'-CGGAATTCGCGGCCGCGCGGTCGGGGGTGCTGGTGG-3'). The PCR conditions were
the same as above. This PCR product was digested with NotI and NcoI restriction endonucleases and cloned into plasmid
p41-6-Bluescript digested with the same enzymes. This created plasmid
p41-N, which contained the 41-6 cDNA with a modified 5' end
containing a NotI site compatible with vector pcDNA3-HA.
This modified cDNA was cloned into pcDNA3-HA as a
NotI /ApaI fragment, creating plasmid pcDNA3-HA-41-6 also called plasmid pcDNA3-HAJBP1. To create
plasmid pEF2-HAJBP1, the plasmid pcDNA3-HAJBP1 was digested with
NotI and AvrII restriction endonucleases and the
fragment containing the insert was cloned into plasmid pEF2-HA digested
with the same enzymes.
The pEF2-ATG-FLAG vector was created by inserting a DNA fragment
encoding the Flag epitope into vector pcDEF3 (56). The DNA fragment
encoding the Flag sequence was created with PCR using the plasmid
pFLR2 (57) as the template and the following primers, 5'-GGGGTACCTATGGACTACAAGGACGACGAT-3' and 5'-GTCTGGCGGATCCGCCTTGTC-3'. The amplified fragment was digested with the BamHI and
KpnI restriction endonucleases and cloned into vector pcDEF3
digested with the same enzymes (56). The plasmid pEF2-ATG-FLAG-N vector
was created by digesting the plasmid pEF2-ATG-FLAG with the
BamHI restriction endonuclease and inserting the
self-annealed oligonucleotide 5'-GATCGCGGCCGC-3' into the digested
BamHI site. The plasmid pEF2-FLAG-JBP1 was created by
digesting the plasmid pEF2-ATG-FLAG-N with the NotI and
XbaI restriction endonucleases and inserting a
NotI/XbaI DNA fragment containing the JBP1
cDNA from the plasmid pBSSK-HA-JBP1. The plasmid pBSSK-HA-JBP1 was
created by digesting the plasmid pcDNA3-HA-JBP1 with the
AseI restriction endonuclease, blunt-ending with the large
fragment of DNA polymerase I (Klenow), and inserting the fragment
containing the HA-JBP1 insert into the plasmid pBluescriptSK (Stratagene) digested with the restriction endonuclease
EcoRV and dephosphorylated with alkaline phosphatase. The
vector pEF2-ATG-Myc was created by inserting a DNA fragment encoding
the Myc epitope into vector pcDEF3 (56). The DNA fragment encoding the
Myc sequence was created with PCR using a plasmid containing the Myc
epitope as the template (a gift from Jerry Langer) and the following
primers, 5'- GGGGTACCATGGAAGAGCAGAAGCTGATC-3' and
5'-CCGGATCCAGGTCCTCCTCAGAGATC-3'. The conditions for PCR were the same
as above. The amplified fragment was digested with the BamHI
and KpnI restriction endonucleases and cloned into vector
pcDEF3 digested with the same enzymes (56). The plasmid pEF2-ATG-Myc-N
vector was created by digesting the plasmid pEF2-ATG-Myc with the
BamHI restriction endonuclease and inserting the
self-annealed oligonucleotide 5'-GATCGCGGCCGC-3' into the digested
BamHI site. JBP1 was cloned into pEF2-ATG-Myc-N as a
NotI/XbaI fragment from pEF2-Flag-JBP1.
Construction of pEF2-HAJBP1D4--
The plasmid pEF2HAJBP1D4 was
created to express a carboxyl-terminal fragment of the JBP1 protein for
purification and deletion analysis. A fragment of the JBP1 cDNA was
amplified by PCR with the plasmid p41-6-Bluescript as the template and
the T7 oligonucleotide and the oligonucleotide 41-del-4
5'-GCGGCCGCGCCCAGCACTTCCTAAAAGATG as primers. The PCR product was
cloned into vector pCR2.1 (Stratagene) and then subcloned as a
NotI/SpeI fragment into vector pEF2HA digested
with NotI and XbaI restriction endonucleases.
Creation of JBP1 Point Mutants--
The PCR was used to create
two point mutants of JBP1 called JBP1R368A and
JBP1G367A. To create JBP1R368A, a fragment of
JBP1 was amplified with the oligonucleotides 41Del-1 (5'-GCGGCCGCCCCTTGGTGGCACCAGAGG-3') and MUT1
(5'-GTTCACCAGGGGTCCCGCTCCTGCTCCC-3'; changed bases shown in
boldface) and the plasmid pEF2-Flag-JBP1 as the template. Thirty cycles
at 94 °C for 40 s, 42 °C for 40 s, and 72 °C for
40 s were performed with the same buffer and concentrations as
mentioned above. To create JBP1G367A, PCR was performed as
described for JBP1R368A except that the oligonucleotide
MUT2 (5'-GTTCACCAGGGGTCCCCGTGCTGCTCCC-3') was used instead
of MUT1. After amplification, these fragments were extracted with
phenol:chloroform:isoamyl alcohol, precipitated with ethanol, and
digested with the restriction endonucleases SanDI and
NdeI. The fragments containing the mutations were purified from agarose and ligated into the plasmid pEF2-Flag-JBP1 digested with
same restriction endonucleases. To express the JBP1R368A
and JBP1G367A proteins fused to the Myc epitope, the
inserts from the Flag constructs were cloned into the pEF2-ATG-Myc-N
vector as NotI/XbaI fragments.
Cell Culture and Transient DNA Transfections--
COS-1 cells
(58), derived from a simian kidney line, were transfected with plasmids
by the DEAE-dextran/Me2SO shock protocol (59-61). Tissue
culture dishes (10-cm dish, Falcon) containing 10 ml of 10% calf
serum-supplemented Dulbecco's modified Eagle's medium (DMEM) were
seeded with 1.75-2.0 × 106 cells trypsinized at
confluence. Cells were incubated overnight at 37 °C before
transfecting. For each dish, 636 µl of DMEM, 160 µl of DEAE-dextran
(10 mg/ml, filter-sterilized), and 4 µl of chloroquine diphosphate
(60 mg/ml, filter-sterilized) were combined. Next, for each dish
transfected, 5 µg of plasmid DNA was diluted in 200 µl of TBS (25 mM HCl, 136 mM NaCl, and 2.7 mM
KCl, pH 7.4) and then added dropwise to the 800-µl
DMEM/DEAE-dextran/chloroquine diphosphate mixture. Medium was aspirated
from a dish of cells, then this DNA/DMEM/DEAE-dextran/chloroquine
diphosphate mixture was then added to the dish, swirled gently for a
few seconds, topped with 3 ml of 2.5% calf serum supplemented DMEM,
and incubated for 3 h at 37 °C. After the incubation, the
medium was removed by aspiration and the cells were shocked by the
addition of 3 ml of PBS containing 10% Me2SO. After 2 min
the Me2SO was aspirated, the cells rinsed with 3 ml of PBS,
and then returned to the 37 °C incubator with 10 ml of complete
DMEM. Cells were harvested for assays after 48-72 h.
Isolation of COS Cell Lysate--
Dishes of adherent COS cells
were washed with ice-cold PBS and scraped with a silicone policeman in
0.5-1.0 ml of ice-cold Lysis Buffer (150 mM NaCl, 50 mM Tris·HCl, pH 8.0, 2.5 mM
MgCl2, 10.3 mM NaF, 1.05 mM
Na3VO4, 5 mM sodium pyrophosphate,
1% IGEPAL (Sigma catalog no. I-3021), 0.8 mM
phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 2 µg/ml
antipain, 10 µg/ml benzamidine, 104
kallikrein-inactivating units/ml aprotinin, 1 µg/ml chymostatin, 1 µg/ml pepstatin). Cells were incubated on ice for 20 min to 12 h
and spun down at 16,000 × g (Eppendorf 5415 microcentrifuge, 14,000 rpm) for 10 min at 4 °C. Supernatants were
transferred to a new 1.5-ml microcentrifuge tube, and lysates were
stored at 70 °C or in a liquid nitrogen freezer until used.
Isolation of HeLa Cell Lines Expressing HA-JBP1--
HeLa cells
were cultured in DMEM supplemented with 10% fetal bovine serum (FBS).
Cells from two T-150 flasks were grown to 90% confluence, and adherent
cells were trypsinized and transferred to a 15-ml tube. Cells were
washed twice with ice-cold PBS and resuspended in 0.45 ml of PBS. Ten
µg of plasmid DNA, pEF2HA, or pEF2HA-JBP1, was added to the cells and
mixed by pipetting. The mixture of cells and DNA was transferred to an
ice-cold cuvette with a gap of 0.4 cm. Cuvettes were pulsed with a
Bio-Rad Gene Pulser II set at 220 V and 950 microfarads. After
electroporation, 1 ml of DMEM containing 10% FBS was added and the
cells were mixed well by pipetting. The contents of the cuvette were
transferred to a 150 mm dish and DMEM containing 10% FBS was added.
After 72 h, cells were grown under selection with 450 µg/ml G418
(Geneticin). Independent colonies were selected and expanded in
separate dishes. Clones were analyzed by Western blotting with anti-HA
antibody for expression of HA-JBP1.
In Vitro Binding Assay--
To express the recombinant protein
fused to glutathione S-transferase (GST), the insert from
plasmid p31-2B was cloned into vector pGEX1 (Amersham Pharmacia
Biotech) as a BamHI/XhoI fragment creating
plasmid pGST-JBP1. This fused amino acids 268-637 of JBP1 to GST and
is referred to as GST-JBP1-N268. This plasmid was transformed into
E. coli strain DH5 F'IQ. Induction and purification of
the GST-JBP1 fusion protein were performed as follows. Cultures of 250 ml were grown to an OD600 of 0.8 at room temperature.
Cultures were induced with 1 mM IPTG overnight at room
temperature. Cells were pelleted by centrifugation at 4700 × g (rotor H6000A, 4,000 rpm) in a Sorvall RC3C centrifuge at
4 °C. Cells were washed one time with Buffer A (150 mM
NaCl, 50 mM Tris·HCl, pH 7.4, and 0.8 mM
PMSF), then repelleted and resuspended in 12.5 ml of Buffer A and
sonicated. Triton X-100 (20%) was added to a final concentration of
1%, and lysates were incubated at 4 °C with agitation for 20 min.
Lysates were centrifuged at 4 °C for 20 min at 8,720 × g (rotor SS-34, 18,000 rpm) in a Sorvall RC5B centrifuge.
Supernatant was divided into 0.8-ml aliquots, frozen in a dry ice
ethanol bath, and stored in liquid nitrogen or at 70 °C. To
perform the in vitro binding assay, 0.8 ml of frozen lysate
was thawed and centrifuged at maximum speed in an Eppendorf 5415 centrifuge for 2 min. The GST control or GST fusion protein
(GST-JBP1-N268) was purified by incubating a fraction of this lysate
(equivalent to 0.4-2.0 µg of fusion protein) with 50 µl of 50%
glutathione-agarose (Sigma no. G4510) in Buffer A for 30 min at 4 °C
with rocking. Afterward, the beads were washed twice in 1 ml of Buffer
B (Buffer A containing 500 mM NaCl) for 5 min. This was
followed by three additional washes in 1 ml of Buffer C (25 mM Tris·HCl, pH 7.4, 50 mM KCl, 10 mM MgCl2, 2% glycerol, 0.8 mM
PMSF, 0.1% Triton X-100, and 100 µg/ml BSA) for 3 min. Buffer C was
added to the beads to yield a final volume of 100 µl.
The binding reaction was carried out by incubating 25 µl of washed
beads in Buffer C with lysate from COS cells overexpressing different
Jak kinases. After incubation for 1 h at 4 °C, the beads were
pelleted for 2 min at 500 × g, then washed twice for 2 min with rocking in 1.0 ml of Buffer C. Bound proteins were eluted by
adding 25 µl of 2× sample buffer (100 mM Tris·HCl, pH
6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 200 mM
DTT) to the washed beads and boiling for 2 min. Eluted proteins were
resolved by SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) membranes (Bio-Rad) with the Trans-Blot SD semidry transfer cell (Bio-Rad, catalog no. 170-3910) according to manufacturer's
instructions. Membranes were rinsed in PBS (138 mM NaCl,
2.7 mM KCl, 1.5 mM KH2PO4, 8.2 mM
Na2HPO4) containing 0.05% Tween 20 and blocked in a solution containing 3% nonfat dry milk (Shoprite brand), 1%
bovine serum albumin, 1 M dextrose, 10% glycerol, 0.05%
Tween 20, and 0.05% thimerosal (Sigma no. T8784) for 1-12 h at room temperature. Membranes were probed with the indicated primary antibodies for 1-12 h and washed twice in PBS (138 mM
NaCl, 2.7 mM KCl, 1.5 mM
KH2PO4, 8.2 mM
Na2HPO4) containing 0.05% Tween 20 for 15 min
per wash; then incubated for 1 h with the indicated secondary
antibody conjugated to horseradish peroxidase followed by washing as
above. The bands were visualized with Western blot Chemiluminescence
Reagent Plus from NEN Life Science Products (catalog no. NEL105).
Immunoprecipitations from Mammalian Cells--
Dishes of
adherent mammalian cells were washed twice with ice-cold PBS and
scraped with a silicone policeman in 0.5 ml of Lysis Buffer. Cells were
incubated on ice for 20 min to 12 h and spun down at 16,000 × g for 10 min at 4 °C. Supernatants were transferred to
a new 1.5-ml microcentrifuge tube. To immunoprecipitate epitope-tagged
JBP1, 0.2 ml of the above lysate was incubated with 0.5-2.0 µg of
the appropriate antibody for 1 to 12 h with rocking. Protein A/G
plus beads (Santa Cruz sc-2003) were added and incubated for 1 h
with rocking. The beads were pelleted and then washed three times in 1 ml of ice-cold Lysis Buffer for 1 to 10 min per wash. Bound proteins
were eluted with sample buffer and resolved with SDS-PAGE. Proteins
were transferred and blotted for Western analysis.
Affinity Purification of Flag-JBP1 and
(His)6-JBP1D4--
Flag-JBP1 was transiently expressed in
COS cells, and cell lysates were prepared as described above for
immunoprecipitation. Cell lysate was passed over an anti-Flag M2
affinity column created by packing 1 ml of anti-Flag M2 affinity gel
(Eastman Kodak Co. catalog no. IB13020) into a Econo-Pac disposable
chromatography column (Bio-Rad catalog no. 732-1010). The column was
first washed in 15 ml of ice-cold glycine buffer containing 0.1 M glycine-HCl, pH 3.5, followed by washing with 15 ml of
TBS (50 mM Tris·HCl, 150 mM NaCl, pH 7.5).
Cell lysate was passed over the column by gravity flow three to four
times, and the column was washed with 50 ml of ice-cold TBS. Flag-JBP1
was eluted in 1-ml fractions in TBS containing 250 µg/ml Flag peptide
(Kodak catalog no. IB13070), and fractions were analyzed by SDS-PAGE
and Coomassie Blue staining.
To purify recombinant protein, the insert from plasmid pEF2HAJBP1D4 was
cloned into the BamHI site of the pRBSII six-histidine vector of the appropriate frame (94, 95). This fused a six-histidine epitope tag to the NH2 terminus of proteins encoded by the
JBP1D4 DNA insert. E. coli carrying the appropriate plasmid
were induced with 1 mM IPTG for 3-12 h at 37 °C. Cells
were pelleted at 4651 × g (4000 rpm) in a RC3C Sorvall
centrifuge with a H600A rotor. Cells were resuspended in denaturing
Lysis Buffer (final pH of 8.0, 50 mM
Na2PO4, 10 mM Tris·Cl, 6 M guanidine HCl, 100 mM NaCl) at 0.5 ml/2 ml of
liquid culture. The lysate was centrifuged at 14,000 rpm (23, 420 × g) in a Sorvall RC5B centrifuge with an SS-34 rotor.
Supernatant was loaded onto a column containing 2 ml of Talon resin
(CLONTECH) prewashed with denaturing lysis buffer. After loading, the column was washed with at least 20 resin volumes of
Wash Buffer (pH 7.0, 8 M urea, 50 mM
NaH2PO4). Protein was eluded with either
Elution Buffer A (pH 5-6, 50 mM
NaH2PO4, 8 M urea, 20 mM Pipes, 100 mM NaCl) or Elution Buffer B (pH
7.0, 8 M urea, 50 mM
NaH2PO4, 100 mM EDTA). Fractions
were analyzed by Bio-Rad protein assay and Coomassie Blue staining
after SDS-PAGE.
UV Cross-linking of [3H]S-Adenosylmethionine to
Flag-JBP1--
One half µg to 2 µg of BSA, CheR (15), Flag-JBP1,
GST, or GST-JBP1-N268 were each incubated with 5.5 µCi of
[3H]S-adenosylmethionine (NEN catalog no.
NET155H) in cross-linking buffer (50 mM Tris·Cl, pH 7.5, 0.1 M NaCl, 2 mM EDTA, 1 mM DTT) in
a total volume of 0.065 ml. Samples were added to 96 well plates and
incubated on ice at a distance of 3.5-5.0 cm from the UV source (Stratalinker 2400). Samples were exposed to two 0.96 joules of UV
irradiation and the reaction stopped by the addition of 30 µl of 3×
SDS-PAGE sample buffer. Samples were stored at 20 °C overnight,
and then proteins were separated by SDS-PAGE and stained with Coomassie
Blue. This was followed by incubation with Entensify reagent (NEN
catalog no. NEF992) and radiography with Kodak Biomax MR film at
70 °C for 7-14 days.
Methylation of Proteins--
HA-JBP1 was immunoprecipitated with
anti-HA antibody from the HeLa-HA-SPMT cell line as described above and
washed three times for 10 min in 0.5 ml of ice-cold Lysis Buffer. As a
control for the immunoprecipitation, HeLa cells transfected with the
parent vector pEF2HA containing no insert were used. This was followed by rinsing the immunoprecipitates two times with Methylation Buffer (50 mM Tris·HCl, pH 7.5, 1 mM EDTA, and 1 mM EGTA) at room temperature. Protein A/G beads with bound
HA-JBP1 were resuspended in 0.04 ml of Methylation Buffer, and either
no additional methyl acceptor was added or 10-100 µg of pooled
histones (Sigma catalog no. H-9250), individual histones separately
(H1, H2A, H2B, H3, or H4 from Roche Molecular Biochemicals; catalog
nos. 223549, 1034740, 223514, 1034758, 223492), cytochrome c
(Sigma catalog no. C-7752), or myelin basic protein (Sigma catalog no.
M1891) was added. To these mixtures, which were placed on ice after
washing with Methylation Buffer, 0.005 ml (2.75 µCi) of
[3H]AdoMet (NEN catalog nos. Net115 or NET115H) was
added. Reactions were incubated at 30 °C for 30 min, and reactions
were stopped by the addition of SDS-sample loading buffer. Samples were
boiled for 2 min and loaded onto 15% SDS-polyacrylamide gels. After
running (70 mV through the stacking gel and 140 mV through the
resolving gel) the gels were stained in Coomassie Blue, destained,
treated with Entensify, and analyzed by radiography.
Northern Analysis--
Multiple tissue Northern blots
(CLONTECH) were probed with labeled DNA sequences
from JBP1. To probe the Northern blots, a 0.7-kb fragment of JBP1 was
labeled by the random hexamer method (53). The 0.7-kb JBP1 fragment was
created by digesting the JBP1 cDNA with the NdeI and
HindIII restriction endonucleases and purifying the fragment
from a 1% agarose gel.
Development of Antisera--
Antisera were developed in rabbits
at Lampire Biological Laboratories (Ottsville, PA). To develop
antibodies against the JBP1 protein, the (His)6-JBP1D4
protein was purified as described in the section on the purification of
JBP1D4 and injected into rabbit 6511. Rabbit 6511 was immunized with 1 mg of the (His)6-JBP1D4 protein in 5 ml of PBS (2.5 ml
injected intradermally along the back and 2.5 ml subcutaneously into
the axilla) on 12/18/97. This was followed on 1/5/98 with 0.52 mg of
(His)6-JBP1D4 in 4.2 ml (or 1 mg in 7 ml on 1/14/98) of
incomplete Freund's adjuvant (0.5 volume injected intradermally along
the back and 0.5 volume subcutaneously into the axilla). These
injections were followed with intramuscular injections into the hind
leg of the (His)6-JBP1D4 protein in incomplete Freund's
adjuvant as follows: 1 mg in 3 ml on 2/9/98, 1 mg in 7 ml on 3/16/98, 1 mg in 4.5 ml on 4/13/98, 0.83 mg in 9 ml on 5/7/98.
Fluorescence in Situ Hybridization (FISH)--
In
situ hybridization of JBP1 to human metaphase chromosomes was
performed using a modification of the previously published technique
(62). A plasmid containing JBP1 was biotin-labeled by nick translation
and hybridized to metaphase spreads of human lymphocytes on glass
slides. The slides were first pretreated with RNase A (Oncor), washed
in 2× SSC, dehydrated, and then denatured at 70 °C in 70%
formamide, 2× SSC immediately prior to hybridization. The
hybridization mixture (15 µl/slide) contained the biotinylated probe
(300 ng/slide) and excess repetitive human DNA (Blockit, Oncor) in
Hybrisol VI (Oncor). After overnight hybridization at 37 °C, slides
were washed at 45 °C in 50% formamide/1× SSC (2 × 10 min),
1× SSC (2 × 5 min), 0.1× SSC (1 × 5 min), and 0.1× SSC
(1 × 5 min) at room temperature. Probe detection was performed by
incubation of the slides with fluorescein isothiocyanate-avidin (Oncor)
for 20 min at 37 °C. Signal amplification was performed by
subsequent incubation with an anti-avidin antibody (Oncor), followed by
incubation with fluorescein isothiocyanate-avidin. The slides were
mounted in the antifade medium (Oncor) containing diamidinophenylindole
and analyzed using a BX60 Olympus fluorescence microscope. Color prints
of the metaphase spreads showing hybridization with JBP1 were obtained
with Cytovision (Applied Imaging). The slides were subsequently
destained and Giemsa-banded (63).
DNA Sequencing--
Plasmid DNA was isolated and sequenced by
the Murdock Molecular Biology Facility (Missoula, MT) or by the
UMDNJ-Robert Wood Johnson Medical School, Department of Molecular
Genetics and Microbiology, DNA Sequencing Facility (Piscataway, NJ).
Determination of Protein Concentration--
Proteins were
quantitated with the Bio-Rad Protein Assay (catalog no. 500-0006) or by
comparing SDS-PAGE samples stained with Coomassie Blue with BSA as the
reference protein.
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RESULTS |
Identification and Cloning of JBP1 with the Yeast Two-hybrid
System--
To screen for Jak2 interacting proteins with the
two-hybrid system, we cloned the murine Jak2 cDNA into vector pAS2
(50) as described under "Materials and Methods." This construct
fused the GAL4 DNA binding domain (GAL4DBD) to amino acids
19-1129 of the murine Jak2 cDNA.
The yeast strain Y190 was cotransformed with the plasmid pAS2-Jak2 and
a HeLa cell library created for use in the two-hybrid system (51).
Yeast strain Y190 contains two reporter genes whose transcription
indicates an interaction between the two GAL4 fusion proteins. We first
selected transformants for their ability to activate the HIS3 reporter
gene by plating onto media lacking histidine. Histidine prototrophs
were selected and subsequently tested for activation of the
lacZ reporter gene. After screening 1.6 × 106 transformants, 143 histidine prototrophs were selected
to assay for -galactosidase activity. Twenty-eight of those assayed
showed evidence of -galactosidase activity. To determine whether the interactions were dependent on expression of the GAL4-Jak2 fusion protein, we performed a mating assay as described previously (50). Transformants activating both reporter genes were cured of the pAS2-Jak2 plasmid by selecting on media containing cycloheximide. The
plasmid pAS2 contains a marker that confers cycloheximide sensitivity
to the yeast strain Y190, which is cycloheximide-resistant (50). We
confirmed loss of the pAS2-Jak2 plasmid by testing the
cycloheximide-resistant colonies for tryptophan auxotrophy. Colonies,
which now contained only the library plasmid, were mated to yeast
strain Y187 expressing "decoy" GAL4 fusion proteins including lamin, CDK2, or SNF1. We also re-tested for the original Jak2 interaction by including yeast expressing the GAL4-Jak2 fusion protein
in this mating assay. Of the 28 clones that exhibited -galactosidase
activity, 10 activated the lacZ reporter gene in the
presence of the GAL4-Jak2 fusion protein, but not in the presence of
the "decoys." Results from one of these mating assays can be seen
in Fig. 1. Library plasmids from these 10 clones were rescued, characterized by restriction endonuclease
digestion, and sequenced.
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Fig. 1.
Specificity of the interaction between Jak2
and JBP1. Specificity of the interaction between Jak2 and JBP1 in
the two-hybrid system. Three independent colonies of yeast strain Y190
containing a library plasmid encoding JBP1 were mated with yeast strain
Y187 containing the pAS2-Jak2 plasmid as well as three "decoy"
plasmids. The decoy control plasmids contained the GAL4 DNA binding
domain (GAL4DBD) fused to lamin, CDK2, or SNF1. Diploids
were grown on selective media and assayed for expression of
-galactosidase with the paper filter assay. Details of the procedure
are described under "Materials and Methods."
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The 10 clones which activated the lacZ reporter gene only in
the presence of the pAS2-Jak2 construct represented four different cDNAs (46). Three of these cDNAs will be described elsewhere. Two of the 10 clones, called 31-2B and 41-3A, represented independent isolates of the same cDNA. The inserts from clones 31-2B and
41-3A were each roughly 1.5 kb. To isolate a longer clone, we used the 41-3A insert as a probe to screen a human M426 cDNA library (54). This led to the isolation of a 2.4-kb cDNA clone, which codes for a
protein of 637 amino acids called JBP1 (for Jak-binding protein 1),
with a predicted molecular mass of 72.4 kDa. The putative full-length
sequence (GenBank accession no. AF167572) was compiled from our
sequence with an additional 97 nucleotides from a sequence tag recently
entered into the GenBank (accession no. AA417623). This added an
additional two amino acids plus some 5'-untranslated region nucleotides
to our cDNA. The open reading frame of the JBP1 cDNA encoded
the amino acid sequence shown in Fig.
2A.
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Fig. 2.
Predicted amino acid sequence of JBP1 and
its homologues. A, The open reading frame of JBP1 and its
homologues are shown. The sequences for the S. pombe protein
Skb1, C. elegans, S. cerevisiae Hsl7p homologues have
been reported previously (47, 48). The murine homologue of JBP1 called
JBP1MM was obtained by translating the murine bach 2 cDNA sequence
(accession no. D86604) and available murine expressed sequence tag
sequences. The D. melanogaster homologue of JBP1 was
obtained from a clone identified and sequenced as detailed under
"Results." The consensus sequence is shown on the bottom
of each panel. Identical amino acids corresponding to the consensus
sequence are shown in black outline with
white lettering. Similar amino acids are shown in
gray outline with white
lettering. Dots represent spaces inserted to
maintain alignment. The sequences for Hsl7p (GenBank accession no.
P38274) and the JBP1 homologue in C. elegans (GenBank
accession no. P46580) are not shown in their entirety. These sequences
can be accessed from the GenBank. Alignment was generated with the
Genetics Computer Group (GCG) (93) pileup software and shading
generated with Boxshade software available via the World Wide Web.
B, comparison of JBP1 and several protein methyltransferases
(or putative methyltransferases). Asterisks and
Roman numerals indicate methyltransferase regions
I, II, and III as described (68, 69). Alignment was generated as above
and the accession numbers are as follows: PRMT1 (rat)
Q63009, PRMT1 (human) Q99873, RMT1 (yeast)
P38074, and PRMT2 (human) P55345. Human and rat
PRMT1 and yeast RMT1 have been shown to have
methyltransferase activity. The gene for PRMT2 has been
localized, but no biochemical activity for its encoded protein has been
reported.
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In Vitro Interaction between GST-JBP1 and the Jak Kinases--
To
determine whether JBP1 could interact with Jak2 outside of the
two-hybrid system, we created a GST fusion protein by cloning the
insert from the plasmid p31-2B into the pGEX1 expression vector through a compatible BamHI site (see "Materials and
Methods"). This fused amino acids 268-637 of JBP1 to GST and is
referred to as GST-JBP1-N268. We then purified and immobilized
recombinant GST-JBP1-N268 onto glutathione-agarose beads. To provide a
source of the Jak proteins, COS-1 cells were transiently
transfected with different Jak kinase expression plasmids. Lysates from
these cells were prepared and incubated with immobilized GST or
GST-JBP1-N268. Proteins that bound to the beads were eluted by boiling
in sample buffer, separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the respective anti-Jak antibodies.
GST-JBP1-N268, but not GST alone, was able to bind murine Jak1, murine
Jak2, murine Jak3, and human Tyk2 from COS-1 cells (Fig.
3). The interaction (with Jak2) did not
appear to require an active kinase domain, as GST-JBP1-N268 was also
able to interact with a kinase inactive mutant of Jak2.
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Fig. 3.
In vitro binding of
GST-JBP1-N268 to the Jak kinases. Lysates from COS-1 cells
expressing different Jak kinases were incubated with the GST-JBP1-N268
(indicated as GST-JBP1 in the figure) fusion protein or GST alone
(0.8-3.0 µg) immobilized on glutathione-agarose. The proteins
associated with GST or GST-JBP1-N268 were resolved with SDS-PAGE,
transferred to PVDF membranes, and blotted with anti-Jak1, anti-Jak2,
anti-Jak3, or anti-Tyk2 antibodies. I represents 10% of the
input COS-1 cell lysate used for the binding assay.
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Identification of Sequence Homology between JBP1 and Protein
Methyltransferases--
Initial searches of the GenBank data bases
with our 41-3A sequence revealed homology to sequence tags from a
variety of different libraries. Subsequent entries led to the
identification of JBP1 as a homologue of the protein encoded by the
S. pombe gene skb1 and the S. cerevisiae gene HSL7 (47, 48). In these reports, homologous open reading frames from an uncharacterized human cDNA sequence were identified (47, 48). Since our original report (46),
additional sequence data for this human cDNA have been entered into
the GenBank data base (accession no. AF015913). This sequence is
identical to our JBP1 sequence with the exception of a substitution of
valine for glycine at position 553 and phenylalanine for serine at
position 247 (Fig. 2). In addition, when we searched the data bases
with our sequence, it matched highly to the complement of the
3'-untranslated region of a murine cDNA encoding bach protein 2 as
well as other murine sequence tags. When these murine sequences were
compiled and translated (GenBank accession no. AF167573), they encoded
a protein with extremely high homology to our human cDNA as shown
in Fig. 2A. We have also obtained a D. melanogaster homologue of JBP1. By searching available data bases,
we identified Drosophila expressed sequence tags with
homology to JBP1. After obtaining these clones (LD07634 and LD08768)
from Genome Systems Inc., they were sequenced with vector and internal
primers (GenBank accession no. AF167574). Initial sequencing revealed
that clone LD08768 had a deletion of amino acids 126-130. Whether this
represents a physiological splice variant of this protein remains to be
seen. Fig. 2A shows the homology between members of the JBP1
family which currently includes sequences or cDNA clones from
S. pombe, S. cerevisiae, Caenorhabditis
elegans, D. melanogaster, Mus musculus, and Homo
sapiens. Since we used the murine Jak2 cDNA as the bait to
screen a human library, we anticipated that any proteins identified would be highly conserved between mice and humans as is the case with
these proteins.
Other than the open reading frames previously reported (47, 48) and the
Drosophila and murine homologues of JBP1 reported here, JBP1
shares little similarity to other known proteins and contains no easily
recognizable domains. However, continued analysis revealed that JBP1
shared some homology to protein arginine methyltransferases (64-67).
Regions that had been described to be conserved in methyltransferases appeared to be present in JBP1 and its homologues (68, 69). These three
regions are shown in Fig. 2B. Because of this homology and the fact
that methyltransferases have been shown to vary widely in their primary
sequence, we determined whether JBP1 exhibited protein
methyltransferase activity (70).
Cross-linking of JBP1 to [3H]AdoMet--
To
determine whether JBP1 represented a new methyltransferase, we first
measured whether this protein could bind to the universal methyl donor
AdoMet. One method that has been frequently employed to determine
AdoMet binding is UV cross-linking (71, 72). To express JBP1 in
mammalian cells, we cloned this sequence into mammalian expression
vectors containing the Flag epitope. This construct fused amino acids
5-637 of JBP1 with the Flag epitope. We purified FLAG-JBP1 from
transiently transfected COS cells by affinity chromatography, incubated
this protein with [3H]AdoMet, and exposed the reaction
mixture to UV light as described under "Materials and Methods." As
a positive control, we used the CheR methyltransferase, a well studied
bacterial methyltransferase whose crystal structure was determined
(70). We included BSA, GST, and GST-JBP1-N268 as additional controls.
Fig. 4A shows that of the four
proteins tested only CheR and Flag-JBP1 were able to be cross-linked to
[3H]AdoMet.
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Fig. 4.
Binding of JBP1 to AdoMet and analysis of
JBP1 methyltransferase activity. A, cross-linking of
Flag-JBP1 to [3H]AdoMet. Left side,
Coomassie Blue-stained gel; right side,
radiography of left side. Proteins (0.2-7.0 µg
of each) were incubated in cross-linking buffer with 5.5 µCi
[3H]AdoMet as described under "Materials and
Methods." Samples were placed into individual wells of a 96-well
microtiter plate on ice and exposed to two pulses of 9600 joules in a
2400 Stratalinker (Stratagene). Afterward, 3× SDS-PAGE sample buffer
was added and samples were stored at 20 °C. To visualize the
cross-linked proteins, samples were separated by SDS-PAGE, stained in
Coomassie Blue, and the radiographic signal amplified with Entensify
(NEN Life Science Products), and dried on Whatman 3M paper. Dried gels
were placed on Kodak Biomax-MR film and stored at 70 °C for 7-18
days. Bars indicate the position of the molecular weight
standards shown on the Coomassie Blue-stained gel. B,
in vitro methyltransferase reactions. Top
panel, Coomassie Blue staining; bottom
panel, radiography of top panel. HA-JBP1 (or HA alone) was
immunoprecipitated from HeLa cells as described under "Materials and
Methods." Immunoprecipitates were washed in lysis buffer, followed by
a wash in methylation buffer. Washed immunoprecipitates were incubated
with [3H]AdoMet alone or [3H]AdoMet plus
12.5 µg of histones (Sigma catalog no. H9250), myelin basic protein
(Sigma catalog no. M1891), or cytochrome c (Sigma catalog
no. C7752) for 30 min at 30 °C. Reactions were stopped by the
addition of 3× SDS-PAGE sample buffer. Proteins were separated by
SDS-PAGE and labeled proteins visualized by radiography after 12-48 h
on Biomax-MR film. Arrows indicate the position of the
molecular weight standards shown on the corresponding Coomassie
Blue-stained gel (above). Arrowhead in the Coomassie
Blue-stained gel shows the JBP1 doublet. C, analysis of
methyltransferase activity in immunoprecipitates of point mutants of
JBP1. Myc-JBP1, Myc-JBP1R368A and Myc-JBP1G367A
were expressed in COS-1 cells and immunoprecipitated as described under
"Materials and Methods." Empty pEF2-Myc vector was used as a
negative control. Immunoprecipitates were incubated with 100 µg of
histones and [3H]AdoMet as in B.
Arrows indicate the position of the molecular weight
standards shown on the corresponding Coomassie Blue-stained gel (to the
left).
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JBP1 Immunoprecipitates Contain Protein Methyltransferase
Activity--
Given that JBP1 had homology to a protein
methyltransferase and was able to be cross-linked to
[3H]AdoMet, we determined whether JBP1 could transfer
labeled methyl groups from [3H]AdoMet to proteins. HeLa
cells were transfected with the plasmid pEF2HA-JBP1, and stable cell
lines expressing HA-JBP1 were isolated. We immunoprecipitated HA-JBP1
from HeLa cells and incubated this protein with histones and
[3H]AdoMet. Histones were selected because they have
previously been shown to function as a methyl acceptor for some protein
methyltransferases (73-75) and because of the genetic link between
HSL7 and histones in yeast (48, 75). As a control we used
immunoprecipitates from HeLa cells transfected with the pEF2-HA vector.
Incubations of histones with [3H]AdoMet or
[3H]AdoMet plus immunoprecipitates from pEF2-HA
vector-transfected HeLa cells resulted in no transfer of the
radioactive methyl groups (Fig. 4B). However, incubation of
histones with [3H]AdoMet plus immunoprecipitates from
transfected cells expressing HA-JBP1 resulted in transfer of methyl
groups from [3H]AdoMet to histones. To determine whether
HA-JBP1 could transfer methyl groups to other methyl acceptors, we also
included two additional known methyl acceptors, cytochrome c
and myelin basic protein (74, 76). Myelin basic protein, but not
cytochrome c, could serve as a methyl acceptor for
HA-JBP1.
In addition to reactions containing the exogenously added methyl
acceptors, we observed labeled proteins in reactions containing only
HA-JBP1 immunoprecipitates. These additional substrates which co-immunoprecipitate with HA-JBP1 vary in molecular weight and may
represent additional physiological substrates of JBP1.
Analysis of JBP1R368A and JBP1G367A Point
Mutants--
While we identified homology between JBP1 and putative
protein methyltransferases and demonstrated cross linking of JBP1 to AdoMet, the possibility of a contaminating enzyme in our
immunoprecipitates could not be excluded. To address this issue, we
created two point mutants of JBP1 and analyzed immunoprecipitates of
these mutants for protein methyltransferase activity. Because of the
highly conserved nature of the GXGRGP motif in JBP1 and its
homologues and the similar GXGXG motif in other
protein methyltransferases (69), we selected this region for mutational
analysis. Both the conserved arginine in JBP1R368A and the
central glycine in JBP1G367A were mutated to alanine
residues. A similar region has been shown to be involved in AdoMet
binding of the HhaI DNA methyltransferase (77). We compared
the activity of the two point mutants with that of the wild type enzyme
after expression in COS cells. While all three proteins were expressed
in COS cells, the Myc-JBP1 protein had the ability to methylate
histones whereas the mutants exhibited little or no activity as shown
in Fig. 4C. Similar quantities of JBP1 and the two mutants
were present in the immunoprecipitates as determined by Coomassie Blue staining.
JBP1 Specifically Methylates Histones H2A and H4--
To determine
which of the five histones could serve as a substrate for JBP1,
methylation reactions were carried out with preparations of individual
histones. HA-JBP1 was immunoprecipitated from HA-JBP1-producing HeLa
cells as described above. Immunoprecipitates were incubated alone or
with 10 µg of pooled histones, histone H1, H2A, H2B, H3, H4, myelin
basic protein, or cytochrome c (Fig.
5). After separation of the proteins by
SDS-PAGE and radiography, it was seen that only histone H2A and H4 were
methylated in this experiment. Bands in other lanes were due to the
fact that the individual histone preparations were not homogeneous and
contained histone H2A or H4 as contaminants. The protein bands
corresponding to histones H1, H2B, and H3 were not radiolabeled. The
radiographic signals present in the lanes containing histones H1, H2B,
and H3 appear to be the same size as histones H2A and H4 (Fig.
5B). These labeled proteins likely represent histones H2A
and H4, which were present within these preparations. In addition,
labeled proteins of larger molecular weight can be seen in the lanes
for histones H2A and H3. These represent other proteins within these
preparations which can serve as substrates for JBP1. As shown
previously in Fig. 4, myelin basic protein, but not cytochrome
c, could also serve as a substrate for JBP1.
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Fig. 5.
Specificity of JBP1-histone methylation.
A, immunoprecipitates of HA-JBP1 were incubated with
[3H]AdoMet, plus 10 µg of mixed histones, individual
histones, myelin basic protein, or cytochrome c as shown.
Proteins were separated with 15% SDS-PAGE and visualized by staining
with Coomassie Blue. Labeled proteins were visualized with radiography
after the gel was treated with Entensify. Top
panel, Coomassie Blue-stained gel. Bottom
panel, radiography of top panel.
B, Coomassie lanes (C) are shown adjacent to the
corresponding radiography lanes (R) from panel
A. All represents the pooled histone lanes,
H2A, H2B, H3, and H4
represent those lanes containing the respective histones.
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HA-JBP1 Interacts with Itself--
The homologue of JBP1 was shown
to form a homodimer in the two-hybrid system (47). In addition, we
observed a doublet in our HA-JBP1 immunoprecipitates after staining
with Coomassie Blue even though blotting with anti-HA antibodies
revealed only a single band (Figs. 4B and
6). To determine whether both of the
bands of this doublet were JBP1, we blotted our HA-JBP1
immunoprecipitates with antisera generated against JBP1. Both bands of
the doublet reacted with this antisera as shown in Fig. 6, suggesting
that the lower band of the doublet represents the endogenous JBP1 that co-immunoprecipitated with HA-JBP1. We confirmed the ability of JBP1 to
bind to itself with an in vitro binding assay (data not shown). In this assay, the GST-JBP1-N268 protein bound the full-length JBP1 protein produced in COS cells. These data indicate that JBP1 forms
homodimeric or multimeric complexes.
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Fig. 6.
Homodimerization of JBP1. HA-JBP1 was
immunoprecipitated from HeLa cells stably transfected with
pEF2-HA-JBP1. Immunoprecipitates were washed and eluted proteins
separated by SDS-PAGE. After proteins were transferred to PVDF
membranes, the membranes were Western blotted with antibodies against
HA or JBP1. After transfer, the remaining proteins were visualized by
staining with Coomassie Blue.
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Northern Analysis of JBP1--
In our initial searches for related
proteins in the GenBank data base, we noticed that there were expressed
sequence tags corresponding to JBP1 from a wide variety of tissues
including those from embryonic and fetal tissue. To determine the
tissue expression pattern and transcript size, we probed two human
multiple tissue Northern blots with the JBP1 sequence. As can be seen
in Fig. 7, the JBP1 mRNA is widely
expressed in human tissues as a major transcript of 2.5 kb.
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Fig. 7.
Northern analysis of JBP1 in human
tissues. Blots containing oligo(dT)-selected mRNA from
multiple human tissues (CLONTECH) were hybridized
with labeled DNA corresponding to the sequence encoding JBP1. After
hybridization and washing, autoradiography was performed which revealed
the presence of a major transcript between 2.4 and 2.6 kb. Molecular
weight standards are indicated on the left of each panel.
The tissues are as follows: H, heart; Br, brain;
Pl, placenta; Lu, lung; Li, liver;
SM, skeletal muscle; K, kidney; Pa,
pancreas; Sp, spleen; Th, thymus; Pr,
prostate; Te, testicles; Ov, ovaries;
SI, small intestine; LI, large intestine;
Le, peripheral blood leukocytes.
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FISH--
A combination of FISH and sequential G-banding was used
to determine the chromosomal localization of JBP1. In situ
hybridization with the biotinylated cDNA probe regionally localized
JBP1 to a medium-sized acrocentric chromosome. Representative results of hybridization of JBP1 to prepared metaphase spreads are shown in
Fig. 8A. Hybridization of JBP1
was specific to the proximal long arm of a group D [13-15] (70)
chromosome in 10 metaphase spreads analyzed by FISH. A combination of
FISH and sequential G-banding of the same metaphase spreads examined
for hybridization further localized JBP1 between bands q11.2-q21 on the
long arm of chromosome 14 (Fig. 8B).
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Fig. 8.
Localization of the JBP1 gene.
Localization of JBP1 to chromosome 14 by FISH. A, metaphase
chromosomes prepared from peripheral blood lymphocytes were hybridized
with a biotin-labeled cDNA probe (JBP1) and visualized with
fluorescein isothiocyanate-avidin. Hybridization signals detected with
JBP1 were specific to the proximal long arm of a group D [human
chromosomes 13-15] chromosome (arrows). B,
composite of chromosome 14 homologues shown in A after
sequential G-banding. Sequential G-banding of the same metaphase spread
shown in A sublocalized JBP1 between bands q11.2-q21 of
chromosome 14. The bracket to the right of the
human chromosome 14 ideogram shows the location of the hybridization
signals detected by FISH with the JBP1 probe.
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DISCUSSION |
We used the yeast two-hybrid system to identify new
Jak2-interacting proteins. One of the proteins identified was a human homologue of the protein encoded by the S. pombe gene
skb1 (shk1 kinase-binding
protein 1) and that encoded by the S. cerevisiae gene,
HSL7 (histone synthetic
lethal 7) (47, 48). While the defects resulting from the
genetic disruption of skb1 and HSL7 implies that
these proteins may have a role in regulating the cell cycle or the
control of cell morphology, no functional motifs or activities for
these proteins were reported (47, 48). Comparison of the human protein
with homologues in S. cerevisiae (48), S. pombe
(47), C. elegans (47), D. melanogaster (this
study), and M. musculus (this study) demonstrate that their
primary sequence has been well conserved as shown in Fig.
2A. Given the conserved nature of these proteins and their
implication in the control of cellular morphology and the cell cycle,
we sought to determine a biochemical activity for this newly identified
human protein.
Sequence analysis of these proteins revealed homology to protein
methyltransferases (Fig. 2B). While the primary sequence of
protein methyltransferases are poorly conserved, three motifs have been
reported to exist among this diverse family of enzymes (68-70). The
homology between the human protein which we have identified, called
JBP1 and protein methyltransferases included these motifs as shown in
Fig. 2B. One of these motifs, called motif I, frequently contains the sequence GXGXG (68, 69). Motif I is
present in many methyltransferases and was shown by crystallography to
be within the AdoMet binding pocket of the HhaI DNA
methyltransferase (68, 69, 77-79). The conserved GXGRGP
region of JBP1 and its homologues likely represents motif I in these
proteins and is followed by regions homologous to motif II and III as
shown in Fig. 2B.
We first assayed the ability of JBP1 to bind the universal methyl group
donor AdoMet with a UVcross-linking assay (Fig. 4A). After
demonstrating that JBP1 could bind AdoMet, we tested whether this
protein could methylate proteins such as histones, cytochrome c, and myelin basic protein, which are commonly used as
methyl group acceptors. HA-JBP1 immunoprecipitates could methylate
histone H2A, histone H4, and myelin basic protein. In addition, we also observed the methylation of proteins that co-immunoprecipitated with HA-JBP1 from HeLa cells. We speculate that these proteins likely
represent some of the endogenous substrates for JBP1. The ability of
JBP1 to bind AdoMet along with the homology between JBP1 and a number
of protein methyltransferases suggests that the methyltransferase
activity present in our JBP1 immunoprecipitates is due to JBP1. To test
this hypothesis further, we created two point mutants containing
substitutions of the central glycine and the arginine of the invariant
GXGRGP region conserved among JBP1 and its homologues.
Immunoprecipitates from both point mutants exhibited little or no
methyltransferase activity, indicating that JBP1 is required for this
activity (Fig. 4C). In addition, the protein
methyltransferase activity is present when JBP1 is purified by affinity
chromatography with an anti-Flag column (data not shown). This purified
material readily methylates histones and appears to have an even higher
activity for myelin basic protein (data not shown). We are currently
unsure if JBP1 is the sole protein responsible for this activity or
whether it is a subunit of a protein methyltransferase complex (for
reviews, see Refs. 80 and 81). Myelin basic protein and histones have
previously been shown to be methylated on arginine residues (64, 65, 74, 82, 83). Based on the homology between JBP1 and protein-arginine methyltransferases and the ability of JBP1 to label proteins known to
be arginine-methylated, we hypothesize that JBP1 and its homologues are
likely to represent a new group of proteins involved in arginine methylation. Based on its ability to methylate myelin basic protein, JBP1 (and its homologues) may represent the first cloned components of
a type II arginine methyltransferase (80, 81). Unlike the large number
of protein kinases which have been described over the past two decades,
it is only recently that cDNAs encoding arginine methyltransferases
have been identified and characterized (64, 65, 75, 84).
It is interesting that homologues of JBP1 in S. pombe and
S. cerevisiae have been linked genetically and or
biochemically to protein kinases. HSL7 disruption in
S. cerevisiae results in G2 arrest, which is
Swe1-dependent (48). Skb1 binds to Shk1 and is associated
with Cdc2 (49). Our work on JBP1 as well as work on its homologues in
yeast suggest that there may be a link between protein kinases and this
conserved family of proteins. While the link between protein kinases
and protein methylation is unclear, protein methylation may add another
mechanism by which complex cellular events are controlled. It is
possible that involvement of JBP1 may be one mechanism through which
the Jaks exert their influence on the cell cycle.
Methyltransferases represent a diverse family of enzymes which can
transfer methyl groups from AdoMet to a variety of substrates including
nucleic acids, small molecules and proteins (85, 86). Protein
methylation reactions can vary with respect to the site of methylation
and the nature of the covalent bond formed. Methylation of the
-amino group of certain NH2-terminal amino acids as well as the N-methylations of histidine, lysine, and arginine
residues are irreversible, and are likely to play a structural role
(70, 85, 86). Other methylations occurring at carboxyl groups are reversible and may therefore be involved in more dynamic processes (86-89). Protein methyltransferases have been implicated in the repair
of damaged proteins, and knockout of one such protein-repair enzyme in
mice results in the accumulation of altered proteins, retardation of
growth, and fatal seizures (80, 86). Despite the fact that protein
methylation has been known for some time, and a role for protein
methylation in cellular signaling has been the subject of a recent
review, the exact role of this modification in many processes is still
not well understood (85, 86, 90). However, some interesting
observations have been recently reported. Arginine methylation was
shown to facilitate the export of certain hnRNPs (heterogeneous nuclear
riboproteins) from the nucleus (91). Another report demonstrated
binding of a protein arginine methyltransferase to the IFNAR1 chain of
the IFN- / receptor complex and experiments with antisense
oligonucleotides provided evidence that this methyltransferase is
involved in mediating the antiproliferative affect of IFN-/ (75).
Recently, a 72-kDa protein, identical to JBP1, was shown to bind to
pICln (I = current, Cl = chloride, n = nucleotide-sensitive), a protein lacking a definitive function that was
proposed to be a cytosolic regulator of a swelling-induced chloride
channel (92). No activity or functional homology was reported for this
72-kDa protein (IBP72 for 72-kDa pICln-binding protein); however, its interaction with a protein speculated to be involved in signaling is
intriguing (92). The sequence homology between JBP1 and its homologues
suggests that these proteins share similar function. Indeed, the human
homologue of Skb1 was shown to functionally complement skb1 in S. pombe and Hsl7p in S. cerevisiae
(49).3 Now that a biochemical
activity has been identified for JBP1, additional experiments can be
designed to help elucidate the role of this protein and its homologues
in cellular signaling, the cell cycle and the control of cell morphology.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of Molecular
Genetics and Microbiology, University of Medicine and Dentistry of New
Jersey, Robert Wood Johnson Medical School, 675 Hoes Ln., Piscataway,
NJ 08854-5635.
