(Received for publication, September 8, 1995)
From the
Complementary DNAs encoding a previously unidentified
phosphoinositide-specific phospholipase C (PLC) isozyme were cloned
from a rat brain cDNA library by the polymerase chain reaction with
degenerate oligonucleotide primers based on sequences common to three
known -type PLC isozymes. The encoded polypeptide contains 772
amino acids (calculated molecular mass, 88,966 daltons) and is similar
in primary structure to
-type PLC isozymes, with overall sequence
identities of 45% to PLC-
1, 72% to PLC-
2, and 47% to
PLC-
3. Thus, the new PLC isozyme was named PLC-
4. Recombinant
PLC-
4 was purified from extracts of HeLa cells that had been
infected with vaccinia virus containing the corresponding cDNA. The
purified protein exhibited an apparent molecular mass of 90 kDa on
SDS-polyacrylamide gels. The specific activity of PLC-
4 and its
dependence on Ca
were similar to those of PLC-
1.
The distribution of PLC-
4 in 16 different rat tissues was studied
by immunoblot analysis with PLC-
4-specific antibodies of fractions
obtained after an enzyme-enrichment procedure. The 90-kDa
immunoreactive protein was detected unambiguously in only eight tissues
and was present at concentrations that were low compared to those of
other major PLC isozymes. A 93-kDa immunoreactive protein was also
prominent in testis but was not detected in the other seven positive
tissues. The 93-kDa enzyme appears to be derived from a splice variant
of the mRNA that encodes the 90-kDa PLC-
4 and contains an
additional 32 amino acids between the X and Y catalytic domains. Splice
variants have not previously been detected for
-type PLC isozymes.
Phosphatidylinositol-specific phospholipase C (PLC) ()plays an important role in receptor-mediated signal
transduction by generating two second messenger molecules, inositol
1,4,5-trisphosphate (IP
) and diacylglycerol, from
phosphatidylinositol 4,5-bisphosphate (PIP
)(1) .
PLC actually comprises a diverse family of enzymes that differ in
structure and tissue distribution(2, 3, 4) .
Nine mammalian enzymes, two Drosophila enzymes, one Dictyostelium enzyme, and one yeast enzyme have to date been
characterized at the cDNA level; all are single polypeptides and can be
divided into three types (
,
, and
) on the basis of size
and amino acid sequence(3, 4) . The
type
includes four mammalian enzymes (PLC-
1, PLC-
2, PLC-
3,
and PLC-
4) and two Drosophila enzymes (PLC-norpA and
PLC-p21), the
type includes two mammalian enzymes (PLC-
1 and
PLC-
2), and the
type includes three mammalian enzymes
(PLC-
1, PLC-
2, and PLC-
3) and the Dictyostelium and yeast enzymes(3, 4) .
The amino acid
sequences of PLC isozymes are relatively variable with the exception of
two well conserved regions, identified as the X (170 amino acid
residues) and Y (
260 residues) domains, that appear to constitute
the catalytic site. The amino acid sequence similarity in the X and Y
domains is
60 and 40%, respectively, among the nine mammalian
enzymes(3, 4) ; the similarity is greater when members
of the same type of PLC are compared. The sequence between the X and Y
domains is short (40-110 residues) in the
- and
-type
isozymes. However, in
-type isozymes, this region is much longer
(
400 residues) and contains two Src homology 2 (SH2) domains,
which bind phosphotyrosine-containing sequences in other proteins, and
one SH3 domain, which interacts with proline-rich sequences in
cytoskeletal proteins(5, 6) . Furthermore, unlike
- and
-type enzymes,
type isozymes have a long
carboxyl-terminal sequence (
450 residues) downstream of the Y
domain.
All mammalian and Drosophila PLCs possess an
amino-terminal region of 300 residues that precedes the X domain
and contains a pleckstrin homology (PH) domain. The PH domain is a
loosely conserved protein module of
100 amino acids and targets
various proteins to the membrane surface by interacting with either the
subunits of G proteins or
PIP
(7, 8) .
The various PLC isoforms
appear to be activated by different receptors through different
mechanisms. Activation of -type enzymes is achieved by
phosphorylation by autophosphorylated tyrosine kinases as a result of
binding to these kinases via the SH2 domains. Isozymes of the
type are activated as a result of binding either to the
subunits
of G
class G proteins, via the long carboxyl-terminal
region, or to G
subunits, probably through the PH
domain(4, 9) . Regulation of
-type enzymes is not
yet understood. Despite the presence of PH domains in
- and
-type enzymes, there is no evidence that these isozymes are
modulated by G
subunits.
As part of our continuing effort
to detect previously unidentified PLC isoforms and gather clues
pointing to a regulatory mechanism for -type PLC enzymes, we
screened a rat brain cDNA library by the polymerase chain reaction
(PCR) with oligonucleotide primers based on the amino acid sequences
conserved in the X and Y domains of
-type isozymes. Here, we now
describe the molecular cloning of a cDNA corresponding to a new
-type PLC, named PLC-
4.
The underlined
sequences at the 5`-end of each oligonucleotide indicate restriction
enzyme cleavage sites (EcoRI and SphI sites for the
forward and reverse primers, respectively) and two nucleotides (CG)
added to facilitate cloning of PCR products. Primers SS and VA were
based on amino acid sequences common to all three types of PLC, whereas
the remaining three primers were based on sequences specific to
-type isozymes.
Three sequential PCR amplifications were
performed with the GeneAmp PCR system 9600 (Perkin Elmer Corp.). A rat
brain cDNA library in the Uni-ZAP XR vector (Stratagene) was used as
the template for the first amplification reaction. The reaction mixture
contained 0.5 µl of library (1 10
plaque-forming units), 50 pmol of each of the primers SS and VA,
0.1 mM dNTPs, 1 unit of native Taq polymerase (Perkin
Elmer Corp.), and the manufacturer's buffer in a final volume of
50 µl. Amplification was performed for one cycle of 5 min at 80
°C, 3 min at 95 °C, 15 s at 45 °C, and 30 s at 72 °C,
followed by 44 cycles of a 15-s denaturating step at 94 °C, a 15-s
annealing step at 45 °C, and a 30-s extension step at 72 °C
(the final extension step was prolonged to 5 min). Although sequences
corresponding to primers SS and VA are present in all three types of
PLC isozymes, the efficiency of amplification of PLC-
sequences
was expected to be low because of the long distance (
1.7 kb)
between the two primer sites. To eliminate products amplified from the
cDNA sequences of PLC-
1 and PLC-
1, two abundant PLC isoforms
in brain, the products of the first amplification were treated with HindIII and AflIII, which cleave a 908-bp product
derived from the PLC-
1 sequence and a 833-bp product derived from
the PLC-
1 sequence, respectively.
The second amplification
reaction was performed with the more internal, type-specific
primers EP and GW and with the restriction enzyme-treated products from
the first amplification reaction as the template. The reaction mixture
contained 0.2 µl of template and the same reaction components as
for the first PCR. DNA amplification was achieved by 35 cycles with
steps similar to those described above, with the exception that
annealing was performed at 50 °C. The products of the second
amplification were subjected to digestion with AflIII, XhoI (which cleaves PLC-
2 sequence), and PstI
(which cleaves PLC-
3 sequence).
The products from the second
amplification reaction were then further amplified with the innermost
-specific primer, IL, and GW. The reaction was performed for 30
cycles under the same conditions as for the second PCR. Amplified DNA
was digested with EcoRI and SphI, and the products
were separated on an 8% polyacrylamide gel. A 426-bp oligonucleotide
was cut and electroeluted from the gel, purified, and ligated into
pUC19 vector. DNA sequencing was performed according to the Taq dye primer cycle sequencing method and Taq dideoxy
terminator cycle sequencing method on an automated DNA sequencer
(Applied Biosystems model 373A). The 426-bp oligonucleotide revealed a
PLC-like sequence that differed from the sequences corresponding to
known PLC isozymes.
For the purification of PLC-4, HeLa cells were grown at 37
°C to a density of 5
10
cells/ml in MEM spinner
medium supplemented with 5% horse serum, infected at a ratio of 10
viruses per cell with the recombinant virus and a vTF7-3
recombinant vaccinia virus that contained the bacterial T7 RNA
polymerase gene, and harvested 2 days after infection(12) .
Cell pellets (80 ml) were washed three times with phosphate-buffered
saline, suspended in 2 volumes of homogenation buffer (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1
mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl
fluoride, leupeptin (10 µg/ml), and aprotinin (10 µg/ml)), and
were disrupted by sonication. The homogenate was centrifuged at 100,000
g for 1 h, and the resulting supernatant (200 ml) was
collected and divided into five 40-ml portions, each of which was
applied to a preparative TSKgel DEAE-5PW HPLC column (21.5
150
mm) that had been equilibrated with 50 mM Tris-HCl (pH 7.4)
containing 1 mM EGTA and 0.1 mM DTT. Proteins were
eluted at a flow rate of 5 ml/min with linear gradients of 0-0.3 M NaCl for 35 min and 0.3-1 M NaCl for 5 min.
Fractions (5 ml) were collected and assayed for PLC as
described(13) . The fractions (49-51 min) corresponding
to the second peak of phosphatidylinositol-hydrolyzing activity from
each column were pooled and concentrated. The subsequent three
chromatography steps on a preparative TSKgel phenyl-5PW column, a
TSKgel heparin-5PW column, and a Mono Q column were performed according
to procedures similar to those previously described(13) .
The third PCR amplification yielded a 426-bp fragment that revealed a PLC-like sequence distinct from those of known PLC isozymes. Removal of the sequences corresponding to the X and Y domains yielded a 312-bp fragment that was used as a probe to screen the rat brain library. Two clones with a 2.7-kb insert were obtained. Complete sequencing of these two clones revealed an open reading frame of 2,316 bp flanked by a 142-bp 5`-untranslated region and a 255-bp 3`-untranslated region including a poly(A) tail. The translational initiation site (ATG) was assigned to the first methionine codon at nt 143 because of the presence of an in-frame stop codon upstream of this methionine and flanking sequences that fulfill the Kozak criteria for initiation(15) . An in-frame translational termination codon (TGA) was present after codon 772. Therefore, we concluded that this new PLC contained 772 amino acids, with a calculated molecular mass of 88,966 daltons (Fig. 1A).
Figure 1:
Amino acid
alignment of PLC-1, PLC-
2, PLC-
3, and PLC-
4. A, comparison of the amino acid sequence of PLC-
4 deduced
from a rat brain cDNA sequence with those of rat
PLC-
1(16) , bovine PLC-
2(17) , and human
PLC-
3 (R. W. Kriz, D. Park, S. G. Rhee, and J. Knopf, unpublished
results). Dashes represent identities with the PLC-
4
sequence, and dots indicate gaps introduced into the sequences
to optimize the alignment. Amino acid residue numbers are shown at the right of each line. The position where 32 and 14
amino acids are inserted in the putative splice variants, ALT-I and
ALT-II, respectively, is shown by
. Double underlines indicate amino acid sequences used for the production of
anti-peptide antibodies (Ab-454 and Ab-759) specific to PLC-
4.
Demarcation of the X and Y domains is indicated. B, linear
representation of the
-type PLC isozymes. Open boxes X and Y denote the regions of
170 and
260 amino
acids, respectively, which constitute the catalytic site of PLC. Shaded box indicates the position of PH domain of
100
amino acids. The numbers above each box refer to the
first and last amino acids. The extent of sequence identity with the
corresponding regions of PLC-
4 is indicated by percentages, and
overall sequence identity is indicated in parentheses.
A comparison of the
deduced amino acid sequence with known PLC sequences revealed that the
predicted protein was similar in primary structure and overall
structural organization to PLC- type isozymes, with overall
sequence identities of 45, 72, and 47% to PLC-
1, PLC-
2, and
PLC-
3, respectively. Thus, the protein encoded by the cloned cDNA
was named PLC-
4 (Fig. 1B).
Figure 2:
Purification of recombinant PLC-4
expressed in HeLa cells. Cytosolic fractions of sonicated cell lysates
from 25 liters of HeLa cell culture were subjected to successive
chromatography on a preparative TSKgel DEAE-5PW column (panel
A), a preparative TSKgel phenyl-5PW column (panel B), a
TSKgel heparin-5PW column (panel C), and a Mono Q column (panel D). PLC activity in column fractions was assayed with
[
H]PI as substrate. Peak fractions from the first
three column steps were subjected to immunoblot analysis with
antibodies to PLC-
4 (insets in panels A, B, and C). Bars above the activity profiles
indicate PLC-containing fractions pooled for the next step
purification. The final purified enzyme was analyzed by
SDS-polyacrylamide gel electrophoresis on an 8% gel and stained with
Coomassie Brilliant Blue (inset in panel D; the
positions of molecular size standards (kDa) are shown on the right).
Because
of the low level of expression, only 200 µg of purified PLC-4
could be obtained from 20 liters of cultured HeLa cells, despite good
recovery yield (overall 25% relative to the DEAE column preparation).
In contrast,
1 mg of purified PLC-
1 was obtained from 8
liters of cultured HeLa cells that had been transfected with the same
virus vector harboring PLC-
1 cDNA. (
)
The catalytic
activities of PLC-1 and PLC-
4 expressed in HeLa cells were
measured with [
H]PIP
at various
concentrations of free Ca
; both enzymes exhibited
similar specific activities and dependence on Ca
(data not shown).
The
resulting PLC-4-enriched fractions were subjected to immunoblot
analysis with affinity-purified monospecific antibodies to PLC-
4
residues 454-464 (Ab-454) or 759-772 (Ab-759) (Fig. 3). The intensity of the 90-kDa band was strongest with
testis and decreased in the order of brain > skeletal muscle >
thyroid gland > stomach > thymus > aorta > heart. No band
was detected unambiguously with normal or regenerating liver, kidney,
prostate, adrenal gland, intestine, pancreas, or lung. In addition to
the 90-kDa band, testis showed a strong band of 93 kDa and a weak band
of 86 kDa. Only the 86-kDa band was detected in spleen.
Figure 3:
Tissue distribution of PLC-4.
PLC-
4 proteins in various rat tissues were enriched by sequential
chromatography of 2 M KCl extracts on a heparin-Sepharose
CL-6B column and a TSKgel heparin-5PW column from which PLC-
4 peak
eluted at 62 min as shown in Fig. 2C. One-fourth of the
pooled peak fractions (61-64) from each tissue was subjected to
immunoblot analysis with rabbit antibodies to PLC-
4, and immune
complexes were detected with alkaline phosphatase-conjugated goat
anti-rabbit IgG. Lanes at both sides contained
prestained molecular size standards mixed with the indicated amounts of
purified PLC-
4 protein.
By comparing
the immunoblot intensities of the samples with those of purified
PLC-4, the amount of PLC-
4 in various tissues was measured
quantitatively. The complex formed between PLC-
4 and antibodies
was visualized with
I-labeled protein A, and the amount
of radioactivity was determined with a PhosphorImager. From the amounts
of protein loaded on the two heparin columns and the SDS gel, it was
deduced that the amount of PLC-
4 expressed in nanograms per
milligram of protein in the KCl extracts was 7.2 for brain, 20 for
testis (combining 90- and 93-kDa forms), 6.4 for skeletal muscle, and
5.6 for thyroid.
Figure 4:
Alternatively spliced forms of PLC-4
mRNA. A, RT-PCR analysis of PLC-
4 mRNA from various rat
tissues. First-strand cDNAs were generated with 100 ng of
poly(A)
RNA from each tissue and an oligo(dT) primer.
PCR was performed with PLC-
4-specific primers, as well as with
-actin-specific primers as a control, and the final products were
separated on a 1.8% agarose gel. PLC-
4 cDNA was also subjected to
PCR amplification. Gel markers with a 100-bp ladder (Research Genetics
and Life Technologies, Inc.) were loaded in the side lanes. B, schematic diagrams of PLC-
4 splice variants. Two
RT-PCR fragments of 330 bp (ALT-I) and 270 bp (ALT-II) were obtained in
addition to the expected 230-bp fragment from testis poly(A)
RNA. Sequencing of these two fragments indicated the presence of
96- and 42-bp inserts between codons 487 and 488 of PLC-
4. The
amino acid sequences deduced from these inserts are indicated. The
regions against which antibodies Ab-ALT-I and Ab-ALT-II were generated
are underlined. C, restriction enzyme digestion
analysis of RT-PCR products containing the entire coding region of
PLC-
4. Amplification was achieved with primers corresponding to
the 5`- and 3`-untranslated regions of PLC-
4 cDNA. The resulting
2.5-kb products were digested with StuI and EcoRI and
separated on a 1.8% agarose gel. Distinct fragments derived from ALT-I
and ALT-II cDNAs in testis as well as that derived from PLC-
4 cDNA
are indicated.
To
determine whether there are any modifications other than the insertions
of 96 and 42 bp, we amplified fragments encompassing the entire coding
region of PLC-4 cDNA by PCR. Two sets of primers based on
sequences located in the 5`- and 3`-untranslated regions were used. The
resulting products were then subjected to restriction enzyme digestion
with StuI and EcoRI, each of which has only one
recognition site in this region and together are expected to generate
three fragments of 1493, 597, and 411 bp from the PLC-
4 cDNA.
Three fragments with these expected sizes were observed in the
restriction reaction mixtures derived from brain and skeletal muscle
poly(A)
RNA and PLC-
4 cDNA (Fig. 4C). As predicted for fragments derived from the
same molecule, the intensity of the three bands was proportional to
their size. However, in the digestion mixture derived from testis
poly(A)
RNA, the 411-bp band was faint, whereas the
1493- and 597-bp bands were strong; two bands of medium intensity were
apparent at positions corresponding to 507 and 453 bp, the sizes
expected for fragments derived from ALT-I and ALT-II cDNA, respectively (Fig. 4C). This result suggests that ALT-I and ALT-II
mRNAs are likely alternatively spliced forms of PLC-
4 mRNA that
differ from the latter only in the 96- and 42-bp inserts.
To
determine whether the 93-kDa testis protein detected by immunoblot
analysis (Fig. 3) is a product derived from such alternative
splicing, we prepared rabbit antibodies (Ab-ALT-I and Ab-ALT-II,
respectively) to the ALT-I-specific sequence KCPMSCLLICVHVLAQA and the
ALT-II sequence KKAPNSIPESILL. The PLC-4-enriched fractions from
brain, skeletal muscle, thyroid gland, and testis were subjected to
immunoblot analysis with a mixture of Ab-454 and Ab-759, with Ab-ALT-I,
or with Ab-ALT-II (Fig. 5). The 90-kDa band was detected by the
mixture of Ab-454 and Ab-759, but not by Ab-ALT-I or Ab-ALT-II, in all
four tissues. The 93-kDa band was recognized by all antibodies and
detected only in testis, suggesting that this protein corresponds to
ALT-I. No band was detected between the 90- and 93-kDa proteins, where
the putative ALT-II protein would be expected. These results indicate
that ALT-II mRNA is not translated or that the translation product
comigrates with ALT-I in the SDS gel. Because the majority
(APNSIPESILL) of the sequence against which Ab-ALT-II was prepared is
also present in ALT-I, Ab-ALT-II also likely reacts with ALT-I.
Therefore, the reactivity of Ab-ALT-II with the 93-kDa band does not
necessarily suggest the presence of ALT-II.
Figure 5:
Immunoblot analysis with antibodies
specific to PLC-4 and its splice variants. Fractions enriched with
PLC-
4 were obtained from the indicated tissues as described in Fig. 3and were subjected to immunoblot analysis with antibodies
to PLC-
4 (mixture of Ab-454 and Ab-759), ALT-I (Ab-ALT-I), or
ALT-II (Ab-ALT-II).
Our results indicate that PLC-4 is expressed at low
concentrations in a limited number of rat tissues. The 90-kDa form was
unambiguously detected by immuoblot analysis in only 8 of 16 tissues
examined, even after enrichment, and the 93-kDa form was detected only
in testis. Brain is one of the rat tissues relatively rich in
PLC-
4, but the enzyme concentration of 7.2 ng per milligram of
crude extract protein is significantly lower than those of PLC-
1
(70 ng/mg), PLC-
1 (140 ng/mg), and PLC-
1 (180 ng/mg). Faint
RT-PCR bands derived from PLC-
4 mRNA were visible in a greater
number of tissues, probably because of the greater sensitivity of this
procedure.
Regenerating liver was examined for PLC-4 expression
because a PLC-
-like enzyme that is not recognized by antibodies to
either PLC-
1 or PLC-
2 was shown to be expressed specifically
during rat liver regeneration (14) and because our immunoblot
analysis showed that PLC-
3 is not the PLC isozyme specific to
regenerating liver.
The present study suggests that
PLC-
4 also is not expressed in regenerating liver.
Like the
other three -type PLCs, PLC-
4 contains a PH domain sequence
at the amino terminus. However, none of the four PLC-
enzymes is
significantly activated by G
subunits.
The PH
domain of PLC-
1 contains a 14-residue sequence (KVKSSSWRRERFYK)
enriched in basic amino acids (net positive charge of 5) that was shown
to form the core of the binding site for the negatively charged
IP
and PIP
and was suggested to tether the
enzyme to membrane surfaces containing PIP
(18) .
The basic amino acid-rich sequence is well conserved in PLC-
4
(KVRTKSWKKLRYFR); indeed, the
4 sequence has a net positive charge
of 7 and thus would be predicted to interact better than PLC-
1
with IP
and PIP
.
Two splice variants of
PLC-4 mRNA, ALT-I and ALT-II mRNAs, were detected by PCR. ALT-I
mRNA encodes the 93-kDa enzyme that contains an additional 32 amino
acids located between the X and Y domains of PLC-
4. It does not
appear that ALT-II mRNA, which would produce an enzyme with an
additional 14 amino acids in the same region between the X and Y
domains, is translated in sufficient quantities to be detected by
immunoblot analysis. PLC-
4 is thus the first example a
-type
PLC that exists in splice variants. Previously identified splice
variants correspond to
-type PLC isozymes including rat
PLC-
1(19) , bovine PLC-
4(20) , Drosophila PLC-p21(21) , and Drosophila PLC-norpA (22) . Examination of the splicing differences in these PLC
isozymes, including rat PLC-
4, reveals that all occur outside of
the X and Y domains (Fig. 6). Rat PLC-
1 and Drosophila PLC-p21 are alternatively spliced in the carboxyl-terminal region
following the Y domain, where the G
-binding site is
located(12, 23) . Splicing differences in Drosophila PLC-norpA variants occur in the PH domain, to which
G
subunits and PIP
might bind. It is thus
possible that the differences among splice variants result in
differences in the ability to interact with signaling components.
Figure 6: Schematic representations of the splice variants of various PLC isozymes. Differences between splice variants are illustrated by solid lines above or below each schematic. Numbers above each schematic represent amino acid positions at boundaries of the splice-variant domains, and those on the right indicate total number of amino acids.
Splice variants of rat PLC-4, like those of bovine
PLC-
4(20) , differ in the region separating the X and Y
domains. This separating region of
-type PLC isozymes plays
important roles by interacting with various signaling components
through its SH2, SH3, and PH domains. Whether the region linking the X
and Y domains of
- and
-type PLC enzymes also interacts with
signaling molecules is not known. However, this region of all mammalian
- and
-type isozymes is rich in acidic amino acids; 20 of 70,
26 of 76, 29 of 137, and 24 of 100 residues are acidic in
1,
2,
3, and
4 isozymes, respectively, and 11 of 50, 19 of
53, 17 of 44, and 17 of 66 residues are acidic in
1,
2,
3, and
4 isozymes, respectively. Unlike the other three
-type PLC isozymes, PLC-
4 contains a high density (12 of 66
residues) of serine and threonine in the region separating the X and Y
domains. All mammalian
-type isoforms with the exception of
PLC-
4 also contain a high density of serine and threonine residues
in this region. Furthermore, as with other splice variants (22) , PLC-
4 and ALT-I exhibit distinct tissue
distributions. Together, these observations suggest that the 90- and
93-kDa PLC-
4 enzymes might be regulated differentially.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16655[GenBank].