(Received for publication, March 7, 1995; and in revised form, June 12, 1995)
From the
The c-Rmil/B-raf proto-oncogene is a member of
the mil/raf family encoding serine/threonine protein
kinases shown to be involved in signal transduction from the membrane
to the nucleus. We isolated from a mouse brain library B-raf cDNAs containing a previously unidentified 36-base pair
alternatively spliced exon located between exons 8 and 9 and,
therefore, designated exon 8b. Human and mouse B-raf mRNAs
also contain the 120-base pair alternatively spliced exon 10 previously
described in the avian c-Rmil gene. Independent splicing of
these two exons, located between the conserved region 2 (CR2) and the
catalytic domain (CR3) gives rise to mRNAs potentially encoding four
distinct proteins. By using specific sera generated against different
portions of B-Raf, we identified at least 10 protein isoforms in adult
mouse tissues. Some isoforms, in the range of 69-72 kDa, are not
recognized by antisera directed against peptides encoded by exons 1 and
2, indicating the existence of B-Raf proteins with two different
NH extremities. The other isoforms, in the range of
79-99 kDa, contain the amino acids encoded by exons 1 and 2, by
either or both of the alternatively spliced exons, and, possibly, by
another unidentified exon. Analysis of B-raf mRNA expression
by reverse transcriptase-polymerase chain reaction and
immunocharacterization of B-Raf proteins in different tissues of the
adult mouse showed a tissue-specific pattern of B-Raf isoforms
expression. Interestingly, isoforms containing amino acids encoded by
exon 10 are specifically expressed in neural tissues. Taken together,
these results suggest that distinct B-Raf proteins could be involved,
in a tissue-specific manner, in signal transduction pathways.
Proto-oncogenes of the mil/raf gene family
encode serine/threonine protein kinases, which act as signal
transducers from membrane-associated receptors to nuclear transcription
factors(1, 2) . Members of the Raf family play a major
role in the regulation of cell proliferation and are also required for
the determination of cell fate during
embryogenesis(3, 4) . The p72/74 Raf-1 protein encoded
by the c-raf/c-mil gene is ubiquitously expressed in
embryonic and adult mouse tissues. This protein has been shown to act
downstream of Ras and upstream of MAP kinase kinase (Mek-1) in
mammalian and invertebrate cells(1) . Raf-1 associates with the
GTP-bound Ras protein, the activated form of
p21. Binding to Ras does not directly activate
Raf-1 but rather serves to recruit this protein to the membrane. This
step is required for Raf-1 activation by an unknown mechanism and
subsequent activation of Mek-1 by
Raf-1(5, 6, 7, 8, 9, 10, 11) .
A-raf, the second member of this family (12) encodes a
67.5-kDa protein and is expressed predominantly in the epididymis and
ovary(13) . Its contribution to the signaling pathway has not
yet been investigated.
The third member of this family was
identified as the c-Rmil gene in avian and B-raf in
mammalian species(14, 15) . Like the Raf-1 protein,
the B-Raf protein can be activated by growth factors, such as the
epidermal growth factor, the basic fibroblast growth factor, and the
nerve growth factor in PC12 cells (16, 17) in a
Ras-dependent manner (18, 19) and by
granulocyte-macrophage colony-stimulating factor, erythropoietin, and
stem cell factor in human and by interleukin-3 in mouse hematopoietic
cell lines(20) . The B-Raf protein acts downstream of
p21 and upstream of Mek during signal
transduction in PC12 cells and appears to be the major Mek kinase in
nerve growth factor-stimulated PC12 cells (18, 19, 21, 22) and in
brain(23, 24) . We also showed that B-Raf interacts in vivo with Mek-1 and phosphorylates this protein on both
serine 218 and 222(25) . Therefore, B-Raf seems to be
homologous to Raf-1 in many aspects of the intracellular signaling
pathway.
However, the c-Rmil/B-raf gene is expressed in a restricted number of tissues as compared with the ubiquitous c-raf/c-mil gene. Thus, high levels of B-raf/Rmil mRNA were detected in neural tissues, testes, and fetal membranes by Northern blot analysis(13, 14, 15, 26) . The number and size of B-raf transcripts, ranging between 2.6 and 12 kilobases, depend on the tissue analyzed(13, 14) , but the identity, structure, and number of B-Raf proteins have not been unequivocally established. We reported the first identification of the Rmil/B-raf gene product in avian neuroretina cells, as a 93.5-kDa protein, which differs from the other Raf proteins by the presence of additional 100 N-terminal amino acids encoded by two exons(27, 28) . These two exons are also present in the human B-raf gene(29) . A B-raf carboxyl-terminal specific antiserum revealed the presence of two proteins of 75 and 77 kDa in mouse testis and brain lysates(30) . An unidentified protein of 95 kDa was also recognized in mouse brain extracts by an antibody directed against the catalytic domain of v-raf(31) . Moreover, in the rat pheochromocytoma PC12 cell line, B-Raf proteins were identified either as a single 95-kDa band by tryptic mapping (17) or as two proteins of 67 and 95 kDa(16) , depending on the cell clones used. Recently, several proteins with apparent molecular weights ranging from 65,000 to 70,000 and 95,000 to 105,000 were detected in mouse brain extracts with a polyclonal antibody specific to the 12 C-terminal amino acids of B-Raf(23) .
We previously reported
the presence in some avian c-Rmil cDNAs of an alternatively
spliced exon of 120 bp ()(exon 10) located upstream of the
kinase domain (27) between exons 9 and 11 according to the
genomic organization of the coding region of the chicken
c-Rmil/B-raf gene(28) . However, the presence
of a similar and of other alternatively spliced exons in mammals
remained to be investigated. Therefore, we undertook a detailed
analysis of the molecular diversity of mammalian B-Raf proteins and of
their tissue distribution in order to gain new insights into the
specific implications of this gene in signal transduction.
We
describe here the molecular cloning and sequencing of several mouse
B-raf cDNAs differing by the presence of two alternatively
spliced exons. We show that the mouse B-raf gene not only
contains the 120-bp alternatively spliced exon 10 previously described
in avian DNA but also another alternatively spliced exon of 36 bp,
located between exons 8 and 9 (exon 8b). The expression pattern of the
various B-raf transcripts in adult mouse tissues was analyzed
by reverse transcriptase-polymerase chain reaction (RT-PCR). We also
identified at least 10 B-Raf protein isoforms in these tissues by using
specific antisera directed against different portions of B-Raf. These
isoforms differed by the presence of the alternatively spliced exons 8b
and 10, by their NH extremities, and possibly by the
presence of other unidentified sequences. Each isoform exhibits a
specific pattern of expression in the adult mouse tissues analyzed,
those containing amino acids encoded by exon 10 being specifically
found in the central nervous system.
300 ng of poly(A) RNAs
from adult mouse brain or 1 µg of mRNA from adult mouse tissues
were reverse transcribed by priming with random hexamers using avian
myeloblastosis virus reverse transcriptase (Amersham Corp.). Locations
of B-raf-specific primers are indicated in Fig. 1, Fig. 2, and Fig. 4. Amplifications were done with the
following primers, the location of which is indicated in the figures:
O
(5`-GAGTCGACCAATTCCACAGCCTTCC), O
(5`-GAGTCGACGAAAAATTCCCAGAAGTGG), O
(5`-GAGTCGACCCTTTGATCCTGTAATTCCAC), and O
(5`-CTGTCGACCTCCATCACCACGAAACC). Southern blots of PCR products,
separated on a 2% agarose gel, were done using standard procedures (32) and hybridized with 5`-end-labeled oligonucleotides,
specific to the exon 8 sequence (O
;
5`-CGACCAGCAGATGAAGATC) or with O
. The B-raf gene
was analyzed by PCR using 100 ng of genomic DNA from NIH3T3 cells as a
template in a standard reaction, with the same couple of
oligonucleotides O
/O
and
O
/O
. PCR products were cloned in the pUC18
vector and partially sequenced using vector-specific primers.
Figure 1:
Identification of
a B-raf alternatively spliced exon of 36 bp (exon 8b). A, partial nucleotide and deduced amino acid sequences of two
B-raf cDNAs obtained from an adult mouse brain cDNA library by
PCR. The locations of exons 8 and 9 are indicated with arrows,
according to the genomic organization of the coding portion of the
c-Rmil gene(28) . Oligonucleotides used in PCR
amplifications are indicated. B, RT-PCR analysis of adult
mouse mRNAs (300 ng) amplified with O/O
(lane 2), with O
/O
(lane3), and with O
/O
(lane4). Lane1, RT-PCR with H
O
as template and O
/O
as primers. Lane5, size markers are indicated in bp. Lanes1-5, 2% agarose gel stained with ethidium bromide. Lanes6-10, PCR products were transferred and
hybridized with the labeled O
oligonucleotide. Using
O
and O
, we amplified two fragments of 157 and
193 nucleotides, which hybridized with an internal exon 8 primer, only
the largest hybridizing with O
. Using 0
and
O
, we amplified a fragment of 156 bp, which hybridized with
O
. Using O
and O
, we obtained a
fragment of 73 bp, which hybridized with O
. C,
cDNA structure and genomic analysis of the mouse B-raf gene
region between exons 8 and 9. 100 ng of DNA from NIH3T3 cells were
amplified under standard conditions with O
/O
and O
/O
, and PCR fragments were subcloned
and sequenced. Exonic sequences are indicated by capitalletters, and intronic sequences are indicated by lowercaseletters.
Figure 2:
Identification of the mammalian B-raf alternatively spliced exon 10. A, schematic
representation of a partial B-raf exon 10-containing cDNA
between exons 9 and 11. Oligonucleotides used for PCR amplification are
indicated, in the upperline for mouse cDNA
amplification and in the lowerline for human cDNA
amplification. The genomic organization was deduced from that of the
chicken c-Rmil gene(27, 28) . B,
RT-PCR amplification of adult mouse B-raf mRNAs. 1 µg of
brain mRNA was reverse-transcribed, and of the first strand (lane2) was amplified between O and O
or O
and O
. The same primers were used
with a control of reverse-transcribed water (lane1).
Size markers are indicated in bp (lane3). Products
were analyzed on a 2% agarose gel. C, RT-PCR amplification of
human B-raf mRNA. RT-PCR was done with water (lane1) or with human mRNAs (lane 2) as template,
using O
/O
or O
/O
primers. Products were analyzed on a 2% agarose gel. D,
comparison of nucleotide sequences of partial cDNAs containing exon 10,
from quail, mouse, and human. The chicken coding sequence corresponds
to that of the genomic DNA(28) . E, comparison of
deduced amino acid sequences from avian Rmil and mammalian B-Raf
proteins. The sequences of exon 10 are overlined.
Figure 4:
Analysis of tissue-specific expression of
the B-raf transcripts by RT-PCR. The structure of the
B-raf transcripts and location of primers used for PCR are
illustrated on the firstline (A). One
µg of total RNA from 14 tissues was reverse-transcribed using
random hexanucleotide primers, and of the first strand cDNA was
amplified with specific primers indicated on lineA.
The amount of RNA and quality of the first strand were verified using
specific primers of the mouse -actin gene (data not shown). Adult
mouse tissues are as follows: total brain (lane2),
cerebral hemispheres (lane3), midbrain (lane4), cerebellum (lane5), cervical
spinal cord (lane6), dorsal spinal cord (lane7), eye (lane8), kidney (lane9), ovary (lane10), testis (lane11), spleen (lane12), thymus (lane13), liver (lane14), muscle (lane15), heart (lane16), lung (lane17). Lane1 is a control with
reverse-transcribed water. Amplifications were done between the
following: the kinase domain, O
/O
, with 25
cycles and hybridized with labeled O
(B); exon 8b,
O
/O
, with 33 cycles and hybridized with labeled
O
(C); exon 10, O
/O
,
with 33 cycles and hybridized with O
(D); exons
8b to 10, O
/O
, with 35 cycles and hybridized
with O
(E); and exons 8 to 11,
O
/O
, with 30 cycles, and hybridized with
O
(F).
Total
cellular RNA preparations were obtained by the single step method using
acidic guanidinium thiocyanate(33) . RT-PCR analysis of mouse
and human exon 10 were done as for the 36-bp exon 8b, except that the
following oligonucleotides were used: O (5`-GAGACCAGGGGTTTCGTG), O
(5`-CATCCGACTTCTGTCCTCC),
O
(5`-CATTCGATTCCTGTCTTCTG), O
(5`-CCAGGCTCAAAATCAAACAC), O
(5`-CCCCCTTGAACCAACTGATG), and O
(5`-GACTTGATTAGAGACCAAGG). RT-PCR analysis of tissue-specific
expression of the different B-raf transcripts was performed
using standard procedures, as described under ``Results,''
using the oligonucleotides described above and O
(5`-CACATTGGATCCGAGATTCAAGTGATGACTGGG). Control of the quality of
first strand synthesis was done using
-actin-specific primers
(Stratagene) as described by the manufacturer.
A rabbit polyclonal antiserum was also raised against the exon 8b-encoded synthetic peptide CEKFLPEVELQDQR conjugated to thyroglobulin (Sigma) through the amino-terminal cysteine residue using maleimidobenzoyl-N-hydroxysylfosuccinimide ester (Pierce) as a coupling reagent.
B-Raf proteins were immunoprecipitated from lysates of COS-1 cells
or adult mouse tissues, using 5 µl of each B-Raf-specific antibody
or 5 µl of rabbit preimmune serum and 50 µl of Pansorbin
(Calbiochem) as a 10% suspension in lysis buffer, and incubated for 2 h
in ice. Bacterial pellets were washed four times in lysis buffer, and
immunoprecipitated proteins were eluted by boiling for 3 min in 40
µl of Laemmli sample buffer, migrated on SDS-polyacrylamide gel
electrophoresis, and transferred to polyvinylidene difluoride blotting
membranes (Immobilon-P, Millipore Corp.). Membranes were rinsed,
saturated with blocking solution(20) , and incubated with B-Raf
antisera diluted 1:1000 to 1:4000 in blocking solution overnight at 4
°C. Immunostaining was performed with horseradish
peroxidase-conjugated anti-rabbit IgG, using the ECL Western blotting reagents (Amersham Corp.), according to the
manufacturer's instructions.
We searched for the presence
of this sequence in adult mouse brain B-raf RNAs using RT-PCR,
as described under ``Experimental Procedures.'' We amplified
two fragments of 157 and 193 bp with oligonucleotides O and
O
specific to exons 8 and 9, respectively (Fig. 1B). Both fragments hybridized with an
oligonucleotide specific to exon 8 (data not shown), but only the
largest one hybridized with the oligonucleotide O
specific
to the 36-bp insertion. Amplifications with specific primers
(O
/O
and O
/O
) confirmed
these results (Fig. 1B). These observations established
the existence of B-raf transcripts containing this 36-bp
additional sequence.
Since this inserted sequence occurred at the
junction between exons 8 and 9, we analyzed the genomic organization of
the mouse B-raf gene in this region by PCR. NIH3T3 DNA was
amplified with either pair of oligonucleotides O and
O
, or O
and O
(Fig. 1C). Sequencing of amplified DNA fragments
showed that this insertion of 36 bp is flanked by 2.9 and 0.3 kilobase
pairs of intronic sequences at its 5`- and 3`-ends, respectively. These
flanking sequences are in good agreement with the consensus for donor
and acceptor splicing sites (39) (Fig. 1C).
Taken together, these results show that the 36-bp insertion found in
mouse B-raf cDNA corresponds to a previously unidentified
alternatively spliced exon of the B-raf gene.
We previously
described an alternatively spliced exon of 120 bp in the avian
c-Rmil gene, located between exons 9 and 11(27) . We
investigated the expression of this exon in adult mouse brain mRNAs, by
RT-PCR. Using two oligonucleotides located in the exons 9
(O) and 11 (O
), we amplified two fragments of
170 and 290 bp (data not shown). The presence of the 120-bp exon was
confirmed by sequencing the 290-bp DNA fragment. Using specific
oligonucleotides of this exon 10 sequence
(O
/O
), we amplified fragments of the expected
size, confirming the presence of this exon in some mouse B-raf mRNAs (Fig. 2B). We also searched for the presence
of exon 10 in total human B-raf RNA purified from a child
brain biopsy. Using oligonucleotides O
/O
and
O
/0
, we amplified two fragments of 167 and
174 bp, respectively, in agreement with the presence of an insertion of
120 bp in human brain RNAs (Fig. 2C). When aligned for
comparison, the nucleotide sequences of chicken, quail, mouse, and
human exons 10 of the c-Rmil/B-raf genes (27, 28) appeared almost identical (Fig. 2D). The avian sequence differed from the human
and mouse sequences by one and three nucleotides, respectively.
However, these changes do not modify the deduced amino acid sequence
encoded by exon 10, which is, therefore, strictly conserved in the
avian and mammalian species (Fig. 2E).
The diversity
of B-raf mRNAs in the region encompassing exons 8b and 10 was
investigated by RT-PCR. Using oligonucleotides O and
O
specific to exons 8 and 11, respectively, and total RNAs
from spinal cord as a template, we amplified four distinct fragments of
300, 336, 420, and 456 bp (Fig. 3A). The 420- and
456-bp fragments hybridized with an oligonucleotide specific to exon
10, whereas the 336- and 456-bp fragments hybridized with a labeled
oligonucleotide specific to exon 8b (data not shown). Thus, the 300-bp
fragment contains exons 8, 9, and 11; the 336-bp fragment contains
exons 8, 8b, 9, and 11; the 420-bp fragment contains exons 8, 9, 10,
and 11; and the 456-bp fragment contains all five exons (Fig. 3A). These results show that the two
alternatively spliced exons are present either together or separately
on the same mRNA, suggesting that the B-raf gene is
transcribed into at least four distinct mRNAs, designated B1 to B4 (Fig. 3B).
Figure 3:
The B-raf gene potentially
encodes four B-Raf isoforms. A, RT-PCR amplification of mouse
brain mRNA between exons 8 and 11. 1 µg of mRNA (lane3) or water (lane2) was
reverse-transcribed and amplified with O/O
primers and analyzed on a 2% agarose gel. Lane3, size markers are indicated in bp. Designation of the
corresponding isoforms is indicated on the right. B,
partial protein structures of the B-Raf isoforms between exons 8 and
11. The size of exons is indicated on the firstline.
Tissue distribution of B-raf mRNAs was first investigated by using oligonucleotides specific to the catalytic domain, since this domain was shown not to be subjected to alternative splicing (Fig. 4B). We found that B-raf gene expression was rather variable, depending on the tissue. High levels of B-raf transcripts were detected in the nervous system, especially in the midbrain and dorsal spinal cord (lanes4 and 7). In the total eyes (lane8), B-raf mRNA is present at an intermediate level, whereas our previous reports showed that this RNA was expressed at a high level in the neuroretina(14) . This could be explained by the fact that the neuroretina constitutes only a very small proportion of this organ. High levels of B-raf were also found in gonads, particularly in testes (lane11), whereas the kidney, spleen, thymus, liver, and heart (lanes9, 13, 12, 14, and 16) contained intermediate levels of these transcripts. Finally, B-raf was barely detectable in the muscle (lane15) where 35 cycles of amplification are necessary to detect it (data not shown).
Analysis of the expression of B-raf transcripts containing exon 8b was done following 33 cycles of amplification (Fig. 4C). We found that this exon is widely expressed, but its distribution differs from that of the catalytic domain. Thus, in the central nervous system, exon 8b is highly expressed in the cerebral hemispheres and cerebellum (lanes3 and 5), whereas its expression is lower in the midbrain and spinal cord (lanes4 and 6). Interestingly, exon 8b is also found at a moderate level in heart, ovaries, testes, and spleen (lanes16, 10, 11, and 12). Exon 10 displayed a more restricted pattern of expression (Fig. 4D). It is very abundant in neural tissues (lanes2-8) in a rather uniform manner and is also detected in the heart and testes (lanes16 and 11, respectively). Analysis of the B4 transcript expression was performed by amplification between a forward oligonucleotide located in the 5`-end of exon 8b and a reverse primer specific to the 3` extremity of exon 10 (Fig. 4E). Expression of this transcript was restricted to parts of the central nervous system, specifically the mesencephalon and metencephalon (lane4). We also detected a weak expression in other neural tissues and in the heart.
Expression of the B1 transcript,
which does not carry an alternatively spliced exon, was analyzed by
RT-PCR (30 cycles) between the forward exon 8-specific primer
(O) and the reverse exon 11-specific primer
(O
) (Fig. 4F). PCR products were
hybridized with an oligonucleotide specific to the constitutively
expressed exon 9. We obtained the expected four fragments corresponding
to B-raf transcripts, which were molecularly characterized. We
observed a slight decrease in the amplification efficiency for the
large fragments corresponding to the B3 and B4 encoded transcripts. The
B1 transcript was widely expressed in adult mouse tissues but was the
unique form detected in some tissues such as the kidney, thymus, liver,
and lung (lanes9, 13, 14, and 17), after 30 cycles of amplification. This transcript was the
predominant one in testes and ovaries (lanes10 and 11), whereas its level was lower than that of the B2 form in
neural tissues and in the heart (lanes2-8 and 15).
Figure 5: Characterization of four anti-B-Raf sera. Top, schematic representation of the B-Raf protein, indicating the positions of the Raf family conserved regions, the two alternatively spliced exons (8b and 10), and the location of peptides used for immunizations. Peptides encoded by exons 1 and 2 and exon 10 were fused to a bacterial MSII polymerase and purified. The 12-amino acid peptide encoded by exon 8b was synthesized and coupled to thyroglobulin, as a carrier, before injection to rabbits. Bottom, summary of the designation of sera and the structure of antigens used for immunization. The properties of each serum to immunoprecipitate (IP) and/or recognize the avian or murine (chimeric constructs) Rmil/B-Raf proteins by Western blotting (WB) are indicated on the right.
The antisera were tested for their ability to recognize the specific isoforms by immunoprecipitation and Western blotting. Therefore, we transfected COS-1 cells with plasmids encoding the different B-Raf isoforms, and we analyzed their expression products 48 h later (Fig. 6). We investigated the ability of these sera to recognize B-Raf proteins in a Western blot analysis by immunoprecipitating cell lysates of transfected COS-1 cells with the IS11 serum and subsequently probing the electrophoresed proteins with each of the immune sera (Fig. 6, A, B, and C). The ability of each serum to immunoprecipitate B-Raf proteins was tested by analyzing the precipitated materials by Western blotting and probing with the IS11 serum (Fig. 6, D, E, and F). The properties of these four polyclonal sera are summarized in Fig. 5.
Figure 6: Characterization of three B-Raf antisera using overexpressed B-Raf isoforms. COS-1 cells were transfected with pSVL vector as control (lanes1), pSVL/c-Rmil A encoding the avian B1 isoform (lanes2), pSVL/c-Rmil A K483M encoding the avian kinase-defective mutant B1 isoform (lanes3), pSVL/c-Rmil B encoding the avian B3 isoform (lanes4), pCP1 encoding the chimeric B2 isoform (lanes5), pCP2 encoding the chimeric kinase-defective mutant B2 isoform (lanes6), and pCP5 encoding the B3 chimeric isoform (lanes7). Expression of the isoforms in transfected COS-1 cells was controlled by immunoprecipitation of cell lysates followed by a Western blot analysis with the same IS11 antibody. A, B, and C, specific recognition of B-Raf isoforms by the three antisera. COS-1 cells were transfected with the indicated plasmids and immunoprecipitated 48 h later with 5 µl of IS11 serum. Immunoprecipitates were analyzed by Western blotting as described under ``Experimental Procedures'' with immune (IS11, 1:4000) or preimmune (PI, 1:4000) IS11 serum (A); immune IS11 (1:4000) or immune IS8b (1:1000) (B); and immune IS11 (1:4000), preimmune IS10 (1:1000), or immune IS10 (1:1000) (C). D, E, and F, immunoprecipitation of B-Raf isoforms. Lysates of transfected COS-1 cells were immunoprecipitated with IS1/2 (10 µl) (D); with IS8b (10 µl), its preimmune serum (PI 10 µl), or IS8b preadsorbed with 10 µg of antigen (IS8b + Ag) (E); and with IS10, preimmune IS10 (PI), or antibodies preadsorbed with 10 µg of antigen (IS10 + Ag) (F). Immunoprecipitates were analyzed by Western blotting with IS11 (1:4000).
Figure 7: Characterization of B-Raf isoforms. Brain (lane1) and liver (lane2) extracts (400 mg) were immunoprecipitated with IS11 serum (A, C, and D), IS11 preadsorbed with its antigen (B), IS10 serum, or its preimmune serum (E and F). Immunoprecipitates were analyzed on SDS-polyacrylamide gels, transferred on polyvinyl membranes, and probed with IS11 (1:4000) (A, B, and E), IS1/2 (1:1000) (C), or IS8b (1:1000) (D and F) sera. Molecular weights (69,000 and 97,000) are indicated on the right of each gel. Blackarrows indicate specific B-Raf proteins; openarrows indicate nonspecific bands. The firstlowerline indicates the serum used for immunoprecipitation; the secondline indicates the serum used for Western blotting.
Figure 8: Tissue-specific expression of B-Raf isoforms. Tissue extracts (brain (A), spinal cord (B), kidney (C), testes (D), thymus (E), spleen (F), liver (G), muscle (H), heart (I), lung (J)) (400 mg) were immunoprecipitated with 5 µl of IS11 serum, and immunoprecipitates were resolved by Western blotting with IS11 (1:4000) (lane1), with IS8b (1:1000) (lane2), and with IS10 (1:1000) (lane3). Exposure time of chemiluminescence was 5 s for neural tissues and 20 s for other tissues. Molecular weights (69,000 and 97,000) are indicated on the right of each panel. Openarrows, asterisks, and opensquares indicate nonspecific bands. Darkarrows indicate specific bands.
In addition, we detected in brain but not in liver extracts
proteins that are specifically recognized by the IS8b or by the IS10
serum. This showed that some B-Raf proteins contain the peptides
encoded by these alternatively spliced exons, thus confirming the
existence of B2 and B3 isoforms (Fig. 7, D and E). Two proteins with apparent molecular weights of 94,000 and
97,000 were immunoprecipitated with the IS10 serum and recognized by
Western blotting with the IS8b serum (Fig. 6F). They
correspond, therefore, to the B4 isoform. That both proteins were also
recognized by the IS1/2 serum (data not shown) rules out the
possibility that the difference in their apparent molecular weights
could be due to an alternative NH extremity and suggests
the existence of an additional alternatively spliced exon(s), as yet
unidentified in B-raf cDNAs. It is also possible that they
could result from post-translational modifications, such as
phosphorylation. These latter isoforms are marked with an asterisk (*).
In summary, the B-raf gene encodes multiple protein
isoforms, which differ by the presence of four alternatively expressed
regions encoded by exons 8b and 10, the two different
NH-terminal extremities, and, possibly, an unidentified
alternatively spliced exon(s). Since these four alternative structures
would potentially generate 16 isoforms, we obviously did not detect all
possible B-Raf combinations. For example, the short NH
extremity was never found associated with the presence of exon
10. Our analysis of B-Raf proteins in liver, in brain, and in other
adult mouse tissues (see below) allowed us to detect only 10 distinct
B-Raf isoforms. Their designations, apparent molecular weights, and
immunologically deduced organizations are summarized in Table 1.
In other tissues, B-Raf proteins were less abundant and their expression pattern was less complex than in neural tissues. In kidney (Fig. 8C), we detected three isoforms, the major one being the short form SF1 of 67 kDa, and the B1 and B1* forms were weakly detected. Thus, the kidney does not appear to express the two alternatively spliced exons 8b and 10. Among the 10 tissues tested, the liver (Fig. 8G) had nearly the same isoform pattern of expression as the kidney, but the SF1 form was less expressed and the B1 and B1* forms were more abundant. Both B1 and B1* isoforms were moderately expressed in the thymus, which appears to be the unique tissue to express only two isoforms.
All other tissues contained B-Raf isoforms carrying the polypeptide encoded by exon 8b. In the spleen (Fig. 8F), we detected three isoforms, the most abundant being a short form of 69 kDa, which was also recognized by the IS8b serum. The two other isoforms were the B1 and B1* proteins. In the testes (Fig. 8D), a weak expression of the SF1 form was also observed. We characterized two isoforms recognized with the IS8b serum, corresponding to the B2 and B2* forms. The B1* protein was also present in this tissue.
The lung (Fig. 8J) expressed moderate levels of B1, B1*, B2, and B2* and also weak levels of the SF1 isoform. The heart (Fig. 8I) is the only non-neural tissue to express a detectable amount of exon 10-containing proteins and also the only one that did not express the unidentified structure (*). However, the major isoform detected in this tissue was the SF1 form. We detected two proteins with the IS10 serum, one of which reacted also with the IS8b serum; thus, they corresponded to the B3 and B4 isoforms, respectively. Interestingly, we could not detect B-Raf proteins in the muscle. Taken together, our results on the expression pattern of the various B-Raf isoforms are in agreement with those obtained by RT-PCR analysis.
Molecular analysis of B-raf transcripts and immunocharacterization of B-Raf proteins allowed us to identify and to characterize at least 10 B-Raf isoforms in adult mouse tissues, each of them exhibiting a particular pattern of distribution. We showed the presence of two alternatively spliced exons and proposed the existence of an additional one. This demonstrates the high degree of complexity in the structure of B-raf gene products, as compared with other protein kinases. Our results also suggest the existence of tissue-specific regulation of alternative splicing and selection of B-Raf isoforms in adult mouse tissues.
It is likely that the 67-kDa B-Raf protein identified in PC12 cells (16, 40) and in brain extracts (23) corresponds to one of the short forms described in this study. However, we did not detect this short form in several PC12 cell line clones used in our laboratory, as also reported by Stephens(17) . This discrepancy may be due to the PC12 cell clones used. Interestingly, these short forms also present a restricted pattern of distribution and appear to represent the major isoform in some tissues, such as the kidney.
Indirect evidence that the
67/69-kDa short forms possess a functional kinase domain is provided by
a convergent set of data. In some PC12 and hematopoietic cell lines (16, 20) in which activation of B-Raf was studied, an
increased phosphorylation and a relative retardation in gel
electrophoretic mobility of the p67/p69 B-Raf protein were detected
after cytokine activation of these cells. Because of the presence of
the 95-kDa protein in these cells, it was not possible to conclude
whether p67/p69 possesses a kinase activity or whether it is a
substrate of p95. We recently showed that a
short isoform was the only B-Raf protein detected in Jurkat cells and
that this protein possesses an intrinsic kinase activity, which
increased after cell stimulation(20) . Moodie et al.
also reported that different B-Raf proteins, with molecular weights
ranging between 65,000 and 105,000, associate with immobilized
p21
-GMP-PNP (23) . Taken together, these results
suggest that the short B-Raf isoforms interact directly with activated
p21
protein and that this interaction apparently does not
require the presence of the first two coding exons. Interestingly, the
B-Raf short forms, which apparently do not contain sequences encoded by
exons 1 and 2, have a structure similar to that of the A-Raf- and
Raf-1-related proteins. Thus, their specific role in signal
transduction remains to be elucidated.
We also identified two alternatively spliced exons, 8b and 10, encoding 12 and 40 amino acids, respectively, which do not present similarity with known protein sequences. Using specific antibodies IS8b and IS10, we identified 5 and 4 isoforms, respectively, containing these sequences and showed that both inserts can be associated on the same protein.
The 36-bp exon 8b, between exons 8 and 9, is located at a position homologous to that of the 7a alternatively spliced exon identified in the c-mil/c-raf gene(41) . This 60-bp c-mil/c-raf exon encodes 20 amino acids, which are not similar to those encoded by exon 8b of the B-raf gene. Interestingly, expression of the 7a c-mil/c-raf exon is restricted to muscle and, to a lesser extent, to brain (42) suggesting that the tissue-specific exons 7a and 8b could have a specific function in signal transduction.
Exon 10, the amino acid sequence of which is conserved between avian and mammalian species, displays the more restricted pattern of expression since it was found expressed at a high level only in the central nervous system and, to a lesser extent, in heart and testes, as determined by RT-PCR. Exon 10-containing isoforms were detected with the IS10 serum in neural tissues and weakly in the heart. We did not detect exon 10-containing proteins in testes, probably because of the lower sensitivity of the immunological assay. It might be that these isoforms are expressed specifically in neural structures of the heart, which would strongly suggest that alternative splicing of this exon takes place preferentially in neural cells.
Exons 8b and 10 are located in the same region, between the CR2 and the CR3 domains of the B-Raf proteins. They are separated only by the 37 nucleotides of exon 9. This region presents a high polymorphism in B-Raf and also, to a lower extent, in Raf-1 proteins and could, therefore, correspond to a variable region in the raf gene family, as reported for calmodulin kinases(43) . Our immunological data suggest that B-Raf proteins contain at least another alternatively expressed structure, which could either correspond to a post-translational modification of the protein or to a sequence encoded by an unidentified alternatively spliced exon. Extensive molecular analysis of B-raf transcripts should help to confirm this hypothesis.
In other tissues, B-raf expression is also complex but quantitatively less important. We observed a relatively elevated level of B-raf transcripts and B-Raf proteins in testes as also reported by Storm et al.(13) , but this expression is weaker than in neural tissues. Our results establish that other tissues express relatively weak amounts of B-raf transcripts and proteins and that expression of this gene is barely detectable in muscle. Moreover, we showed that the pattern of isoform expression is specific to each tissue.
Thus, the variable region
(between CR2 and CR3), which displays low similarity among members of
the Raf family, could direct interactions of B-Raf isoforms with
specific effectors. There have been only few reports showing that the
presence of an alternatively spliced exon could modify the properties
of a kinase. The presence of a neurospecific exon in the c-Src protein
increases its specific kinase activity(47) . In the tyrosine
kinase receptor TrkC, insertion of alternatively spliced exons results
in the loss of its ability to phosphorylate phospholipase C and
phosphatidylinositol 3-kinase (48) . In calmodulin kinase, an
alternative splicing introduces a nuclear localization signal that
targets a calmodulin kinase isoform to the nucleus(43) .
Therefore, it is possible that the presence of alternatively spliced
exons could modulate the B-Raf kinase activity, the specificity of its
substrates, or its targeting within the cell.
It is not clear whether the B-Raf proteins are implicated in differentiating or proliferating signal transduction pathways. It is interesting that, in testes, B-raf transcripts are detected only in pachytene spermatocytes and more abundantly in postmeiotic spermatids as shown by insitu hybridization (26) . We also detected B-Raf proteins more abundantly in neural tissues, where the vast majority of cells are postmitotic. These results suggest that B-Raf proteins could be involved in differentiation rather than in proliferation processes.