(Received for publication, March 6, 1997, and in revised form, May 19, 1997)
From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616
Two distinct species of the thermostable
inhibitor of the cAMP-dependent protein kinase, PKI and
PKI
, exist that are the products of separate genes. The PKI
form,
as first isolated from rat testis, is a 70-amino acid protein, but the
genomic sequence suggested that an alternate form might exist, arising
as a consequence of alternate translational initiation. This species,
now termed PKI
-78, has been synthesized by bacterial expression,
demonstrated to be equipotent with PKI
-70, and also now demonstrated
to occur in vivo. By Western blot analyses, six additional
species of PKI
are also evident in tissues. Two of these represent
the phospho forms of PKI
-78 and PKI
-70. The other four represent
phospho and dephospho forms of two higher molecular mass PKI
species. These latter forms are currently termed PKI
-X and PKI
-Y,
awaiting the full elucidation of their molecular identity. In adult rat testis and cerebellum, PKI
-70, PKI
-X, and PKI
-Y constitute 39, 23, and 32% and 15, 29, and 54% of the total tissue levels, respectively. In adult rat testis, 35-42% of each of these three species is present as a monophospho form, whereas no phosphorylation of
them is evident in cerebellum. PKI
-78 is present at much lower levels in both rat testis and cerebellum (~6 and 2% of the total, respectively) and almost entirely as a monophospho species. PKI
-78, like PKI
-70, is a high affinity and specific inhibitor of the cAMP-dependent protein kinase. PKI
-Y and PKI
-X, in
contrast, also significantly inhibit the cGMP-dependent
protein kinase.
The cAMP-dependent protein kinase
(PKA)1 plays a central role in the signal
transduction of many hormones. The thermostable protein kinase
inhibitor (PKI) is a highly specific competitive inhibitor of this
enzyme that binds with high affinity to the protein substrate-binding
site of the kinase (1). Its role as a regulator of PKA remains to be
fully identified and appears to include not only the reduction of
protein kinase activity, but also the trafficking of the protein kinase
catalytic subunit between subcellular sites (2-4). Two genetically
distinct forms of PKI ( and
) have been shown to be present in
many tissues (5-9). Both forms act as specific pseudosubstrate
inhibitors of PKA, have similar Ki values in the
subnanomolar range, and are likely both involved in intracellular
trafficking (4). PKI
and PKI
share ~40% identity at the amino
acid level (5), share very closely the same recognition determinants
for the catalytic subunit of PKA (5, 10) and for nuclear export (9),
but are notably distinct in the C-terminal half of the molecule. PKI
and PKI
each have a distinct tissue distribution (6).
The PKI form was first identified in rat testis (5) in a follow-up
of observations made earlier by Means and co-workers (11, 12). The
PKI
form purified was one of what appeared to be multiple forms of
the protein present in testis, as was apparent from the DEAE
chromatographic profile of the partially purified protein. The form
purified was shown by protein sequencing and amino acid analysis to be
a 70-amino acid protein with a blocked amino terminus (5).
Identification of the gene for this protein indicated, however, that in
addition to the appropriate in-frame ATG codon, a second upstream
in-frame ATG codon was present. If translated, this alternate in-frame
ATG codon would give rise to an alternate translational product with a
7- or 8-residue amino-terminal extension with the additional sequence
of MRTDSSEM-, with the specific size dependent upon whether or not the
N-terminal methionine was processed. Translational initiation in
eukaryotes is affected by the context of the nucleotides surrounding
the AUG codon of the mRNA (13). The first and second in-frame AUG
initiation codons in the PKI
gene have the nucleotide sequences
3GUGAUGA+4 and
3GAGAUGA+4. With a G at position
3 and an A at position +4, both of these in-frame AUG codons have
initiation sequences that would readily allow translation to be
initiated at either site (13). Neither, however, has the optimum Kozak
sequence (
3(A/G)CCAUGG+4), and so
read-through of the first upstream AUG codon, to allow initiation of
translation at the second, appeared to be a significant possibility
(see Fig. 1). The sequence of the amino-terminal amino acids of the
isolated 70-amino acid PKI
is consistent with its being directly
synthesized and then processed to remove the terminal methionine and to
add a blocking group (14-16). In contrast, if synthesized from the
first in-frame AUG codon, the amino-terminal sequence context is such
that processing of its methionine would be much less likely to occur.
This paper addresses the existence of this alternate translational
product and also the other forms of PKI
apparent from the DEAE
profile. In total, it is now demonstrated that there exist at least
eight isoforms of PKI
, and in the accompanying paper (17), a ninth
form has also been elucidated. The full nomenclature for these
different species is developed subsequently in this paper. The initial
70-amino acid form isolated from rat testis, and originally termed
PKI
, is now more precisely termed PKI
-70. The form that would be
obtained by processing at the first upstream ATG site is termed
PKI
-78.
Each of the
coding sequences for PKI-70 and PKI
-78 were subcloned into the
Escherichia coli expression vector pET5a by engineering NdeI and BglII sites at the 5
- and 3
-ends of
their coding sequences, respectively, using the polymerase chain
reaction. The vectors were then transfected into the BL21(DE3) strain
of E. coli, and proteins were expressed as described by
Studier et al. (18). Each of these proteins was purified to
homogeneity essentially as described by Van Patten et al.
(5). In brief, for the purification of expressed PKI
-78, bacteria
from 3 liters of suspension (A260 = 0.8) were
collected by centrifugation; lysed with a French press; and suspended
in 25 ml of 10 mM Tris-Cl, pH 7.5, 10 mM NaCl,
and 1 mM EDTA. The supernatant obtained following
centrifugation was heated at 92 °C for 10 min and then centrifuged.
The supernatant from this heat treatment was adjusted to pH 5.0 and
applied to a DEAE ion-exchange column using the conditions for
chromatography and elution as described by Van Patten et al.
(5). Pooled fractions with PKI activity were subsequently purified to
homogeneity by HPLC employing a Nova-Pak reverse-phase C18
column and elution with an acetonitrile gradient in trifluoroacetic
acid as described by Van Patten et al. (5). Purification of
expressed PKI
-70 was accomplished exactly as described for expressed
PKI
-78, except that the DEAE column was at pH 6.0, and the final
HPLC was replaced by gel filtration using a Superdex 75 HPLC column and
elution with 0.3 M ammonium acetate, pH 7.0. The two
protein products were characterized by amino acid analysis and by
determination of the sequence of at least the first 10 amino-terminal
residues. The bacterial expression system for a third putative form of
PKI
, termed
2 by Scarpetta and Uhler (8) and containing the
sequence of PKI
-78 plus an additional 14-amino acid amino-terminal
extension, was also created, and the expressed protein was purified by
similar procedures; however, no evidence for its existence in rat
tissue was observed in these studies.
For Western blot analyses of PKI isoforms, the tissues from adult Harlan Sprague Dawley rats were freeze-clamped with Wollenberger clamps precooled in liquid nitrogen immediately after dissection. The frozen tissue was powdered and then rapidly homogenized with a glass-Teflon homogenizer in 1 mM EDTA, pH 7.0 (1 ml/g of tissue), containing a mixture of protease inhibitors (0.5 mM (2-aminoethyl)benzenesulfonyl fluoride, 1 µM leupeptin, 2 mM benzamidine, 0.1 mM L-1-tosylamido-2-phenylethyl chloromethyl ketone, and 20 milliunits/ml aprotinin). The extracts were heat-treated to 92 °C for 10 min and spun at high speed in a microcentrifuge at 4 °C for 2 min, and the supernatants were used for gel electrophoresis. Alkaline phosphatase treatment, where noted, was accomplished by incubating the tissue extract with 5 units/µl alkaline phosphatase (calf intestinal alkaline phosphatase, Pharmacia Biotech Inc.) in 50 mM Tris-Cl, pH 8.0, and 0.125 mM EDTA plus the same mixture of protease inhibitors at 1:4 dilution for 2 h at 30 °C. After treatment, the samples were directly applied to the isoelectric focusing tube gels.
One- and Two-dimensional Electrophoresis and Procedures for Western Blot Analyses of PKI IsoformsTissue samples for PKI
isoform analyses were resolved by one- or two-dimensional gel
electrophoresis, as noted. One-dimensional SDS-PAGE was in accord with
Laemmli (19) using 12-18% gradient gels in 0.1% SDS and 0.375 mM Tris-Cl, pH 8.8. The first dimension for two-dimensional
electrophoresis utilized 1.5 mm x 7.5 cm tube gels (Hoeffer Scientific
Instruments) and 2% pH 4-6 ampholytes (BDH), with anode and cathode
buffers of 0.085% phosphoric acid and 20 mM sodium
hydroxide, respectively. Following the first dimension, gels were
equilibrated for 15 min in 0.125 M Tris-Cl, pH 6.8, 10%
glycerol, 4.9 mM dithiothreitol, and 2% SDS and then applied to a 12-18% SDS-polyacrylamide gel. For Western blot
analyses, following electrophoresis, the gels were equilibrated in 10 mM CAPS, pH 11.0, for 30 min, and the proteins then
electrophoretically transferred to polyvinylidene difluoride membranes
(Gelman Instrument Co.) for 1.5 h at 25 V. The membranes were
incubated for 1 h in blocking buffer consisting of 0.5% casein
and 0.2% Tween 20 in Tris-buffered saline (150 mM NaCl and
10 mM Tris-Cl, pH 7.4). The polyvinylidene difluoride
membrane was incubated overnight with the designated primary
anti-peptide antibody (1:5000 dilution in blocking buffer) and then
washed 1 × 1 min and 3 × 10 min with Tris-buffered saline
containing 0.1% casein and 0.2% Tween 20. The membranes were then
incubated for 2 h with biotinylated anti-guinea pig donkey IgG
antibody (1:2500 dilution in blocking buffer); washed as before;
incubated for 30 min with alkaline phosphatase-conjugated streptavidin;
washed sequentially with Tris-buffered saline containing 0.1% casein
and 0.2% Tween 20 (1 × 1 min and 2 × 10 min) and with 0.1 M Tris-Cl, pH 9.0, 100 mM NaCl, and 5 mM MgCl2 (5 min); and then developed with the
alkaline phosphatase nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate kit (Pierce 34041). For the SDS gels, molecular weight standards were as denoted in Fig. 2. The relative isoform composition was determined by scanning the Western blot two-dimensional gels and integrating the signals using an IS-1000 digital imaging system (Innotech Scientific Corp.).
Preparation of Polyclonal Anti-peptide Antibodies
Anti-peptide antibodies were prepared by D. Harrison
(of this laboratory) using ovalbumin-peptide conjugates as the antigen. For the anti-PKI-(5-22) and anti-PKI
-(5-22) antibodies, the conjugates were synthesized by derivatization of the amino terminus of
the synthesized peptide with the heterobifunctional cross-linker maleimidohexanoyl-N-hydroxysuccinimide ester and then
cross-linking to N-succinimidyl
S-acetylthioacetate-derivatized ovalbumin (20, 21). For the
anti-PKI
-(60-70) antibodies, the peptide was synthesized with an
additional carboxyl-terminal cysteine, and the conjugates were
synthesized by cross-linking
maleimidohexanoyl-N-hydroxysuccinimide ester-derivatized
ovalbumin to the peptide via the terminal cysteine. All anti-peptide
antibodies were raised in guinea pigs using procedures in accord with
the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. Peptide synthesis and purification were in accord
with previously described methods (10). Chemicals were from Pierce.
Routine assays of PKI inhibitory activity were
as described previously (22) using histone II-A as protein substrate
and the PKA C subunit obtained by bacterial expression (23).
Ki values were determined by the method of Henderson
(24) using 8 µM ATP; Kemptide concentrations of 5.6, 2.8, 1.4 and 0.7 µM; PKI
isoform concentrations from 7.9 to
26.6 nM; 0.8 nM PKA; and all other conditions
as described previously (25). Other kinetic assays utilized 30 µM Kemptide and 0.125 mM ATP. PKG-1
was
kindly provided by Dr. Sharon Francis (Department of Molecular
Physiology and Biophysics, Vanderbilt University). The assay of PKG
inhibitory activity was undertaken as described previously (26) using
[Ala34]H2B-(29-35) as substrate.
Both PKI-78 and PKI
-70 were readily obtained by
the creation of suitable bacterial expression vectors and were each
purified to homogeneity. Their identity was established by amino acid
analyses and amino-terminal sequencing. Their purity was established by SDS gel electrophoresis. We have previously demonstrated that, like the
original PKI
, PKI
-70, as directly isolated from rat testis, is a
specific inhibitor of PKA (5). The expressed PKI
-78 and also the
bacterially expressed PKI
-70 were likewise specific high affinity
inhibitors of PKA and did not inhibit even the highly homologous
cGMP-dependent protein kinase (Fig. 1).
Ki values for all of these forms, as determined by
Henderson analysis (24), were indistinguishable (expressed PKI
-78,
0.14 nM; expressed PKI
-70, 0.1 nM; and rat
testis PKI
-70, 0.14 nM). Each were competitive inhibitors versus protein substrate.
The availability of a purified preparation of PKI-78
as a standard allowed us to address the issue of whether this possible alternate translational initiation form of PKI
occurred
physiologically. Other forms of testis PKI also appeared to be a
likelihood as evidenced by the earlier characterization of PKI by Mean
and co-workers and antibody identification of multiple size forms (11,
12, 27). To make an evaluation of the physiological presence of PKI
-78 and to expand the context of investigation to an assessment of the potential existence of other PKI
forms, specific anti-peptide antibodies were prepared against the peptides PKI
-(5-22)
(TTYADFIASGRTGRRNAI), PKI
-(5-22) (SVISSFASSARAGRRNAL), and
PKI
-(60-70) (GQPKKPLDEDK). The nomenclature for these peptides, as
used previously (5, 10), is based upon the sequence of the full PKI
proteins. The first two of these peptides contain the pseudosubstrate
regions of PKI
and PKI
, respectively, and whereas some degree of
overlapping antigenic specificity might have been expected between
them, as shown below, it was not observed. The third peptide is from a region of PKI
in which there is absolutely no homology to
PKI
.
Western blot analyses with these anti-peptide antibodies revealed a
complex pattern of PKI isoforms. In both testis and cerebellum, the
prominent if not exclusive form of PKI detected is the 75-amino acid
species of PKI that is the form of inhibitor protein originally and
extensively characterized from skeletal muscle and other tissues (1).
This form is readily detected by anti-PKI
-(5-22) antisera, but not
by anti-PKI
-(5-22), anti-PKI
-(60-70), or control sera (Fig.
2). This result demonstrates the specificity of the
anti-PKI
peptide antisera. In cerebellum (Fig. 2), as has also been
seen in some other tissues (data not shown), a small amount of a
slightly lower molecular mass PKI
species is also evident. This
latter form may be related to the PKI-I
species that has been
previously described (28, 29). Western blot analyses of a variety of rat tissues showed the expected profile of distribution of PKI
, with
a high predominance in brain and various muscle types (data not shown).
These data were as anticipated from previous studies based upon
mRNA distribution (6).
The pattern with PKI is considerably more complex than is observed
for PKI
. Western blot analyses and one-dimensional SDS-PAGE showed
the presence of at least three major PKI
size forms of ~7, 14, and
15.5 kDa in both testis and cerebellum (Fig. 2). These three forms were
detected with both the anti-PKI
-(5-22) and anti-PKI
-(60-70) antisera, indicating that all three contained, as a minimum, the homologous amino acid sequences matching both the amino- and
carboxyl-terminal sequences of PKI
-70. These forms were not detected
with either control or anti-PKI
-(5-22) antisera (Fig. 2),
indicating the specificity of the anti-PKI
antibodies, and this set
of data demonstrates the absence of cross-reactivity of either the
PKI
forms with the PKI
antibodies or vice versa. The sizes of
these three forms identified here are closely similar to those of the testis PKI species previously identified by Means and co-workers (11,
27). A fourth low abundance form of PKI
, of slightly higher apparent
molecular mass than the 7-kDa form, was also evident on the
one-dimensional gels from testis and cerebellum (Fig. 2).
Western blot analyses of two-dimensional gels (first dimension,
isoelectric focusing; and second dimension, SDS-PAGE) showed that the
pattern of PKI isoforms is even more complex. In testis extracts, at
least eight forms of PKI
are evident, with each of the three
predominant forms and the fourth less abundant species, as separated
based upon size (Fig. 2), being composed of a pair of charge isoforms
(Fig. 3a). In cerebellum, only the more
cationic species of each pair was evident, except for the low abundance form that was present in both tissues as predominantly the more anionic
species (Fig. 3b). There was a good correspondence between the gel migration of the cationic form of the three most abundant species in testis extracts with their counterparts in cerebellum, suggesting that these forms were likely identical in both two tissues.
The diversity of forms in both testis and cerebellum was evident with
both the anti-PKI
-(5-22) and anti-PKI
-(60-70) antisera (Fig. 3,
c and d) with a matching intensity of the spots. This is indicative that all of the species identified in testis and
cerebellum contain key components (if not all) of the sequence that has
been established for PKI
-70.
The nomenclature for these different forms, as used in this report, is
presented in Fig. 4a. The basis for this
nomenclature is as follows. The spot specified as PKI-70 (Fig.
4a) is identified as such based upon its comigration with
exogenously added purified PKI
-70 as isolated directly from rat
testis (Fig. 3, e and f). This experiment
compares the migration of the form in testis extracts with PKI
-70
purified to homogeneity from rat testis (5). Bacterially expressed
PKI
-70 migrates slightly cationic to this form due to the presence
of one additional charge contributed by the free (unblocked) amino
terminus (data not shown).
The spot specified as PKI-78 (Fig. 4a) is identified as
such based upon its comigration with exogenously added, bacterially expressed, purified PKI
-78 (Fig. 3, g and h).
Given this comigration, it would appear most likely that during the
in vivo synthesis of PKI
-78, there is no processing of
its amino terminus by the cleavage of the amino-terminal methionine and
the addition of an amino-terminal blocking group. The absence of any
processing of the amino terminus of PKI
-78 is as might be predicted
from its amino-terminal sequence (MRTD ... ), which is unfavorable
for such amino-terminal processing (15, 16). The location of PKI
-78 on the two-dimensional gel, in comparison with that of PKI
-70, is
consistent with the difference between them. PKI
-78 is 8 amino acids
greater in size and differs in charge by +1. These data demonstrate
that alternate translational initiation does occur under physiological
conditions and that the PKI
transcript can give rise to both
PKI
-78 and PKI
-70. The level of PKI
-78 (dephospho) seen in
tissues has varied from tissue preparation to preparation and has often
been quite low or not detectable. The relative degree of alternate
translational initiation is discussed subsequently in this paper
following discussion of its phosphorylated form.
Two spots on the two-dimensional Western blot are designated as
PKI-X and PKI
-Y (Fig. 4a). These two forms of PKI
are detected by both the anti-PKI
-(5-22) and anti-PKI
-(60-70)
antisera and are therefore homologous to PKI
-70 in at least these
two regions. As evidenced below, these forms also exhibit high affinity
PKA inhibitory activity. Based upon their degree of migration on
SDS-PAGE, these two forms are apparently of higher molecular mass than
either PKI
-70 or PKI
-78, although in this molecular mass range,
such determinations based upon SDS-PAGE migration are not particularly reliable. Scarpetta and Uhler (8) have suggested the possible occurrence of a higher molecular mass form of PKI
derived by alternate RNA splicing that is identical to PKI
-78, but with a
14-amino acid amino-terminal extension. We have synthesized this form
by bacterial expression. On one-dimensional gels, this form migrates
quite distinctively from either PKI
-X or PKI
-Y at a position
intermediate between PKI
-78 and PKI
-X. PKI
-X and/or PKI
-Y
is therefore not this species, except for the possibility that it might
be derivatized with a significantly sized covalent modification.
In testis extracts, PKI-70, PKI
-78, PKI
-X, and PKI
-Y each
have a more anionic companion species. Treatment of the testis extract
with alkaline phosphatase prompted a marked reduction in the anionic
companion species of PKI
-70, PKI
-X, and PKI
-Y, accompanied by
an increased intensity of the more cationic forms (Fig.
5, upper panels). A similar result was also
obtained using protein phosphatase 2A (data not shown). These data
provide evidence that these anionic forms represent phosphorylated
species, i.e. phospho-PKI
-70, phospho-PKI
-X, and
phospho-PKI
-Y, respectively. The anionic companion form of PKI
-78
was notably less sensitive to alkaline phosphatase treatment (Fig. 5,
upper right panel). To evaluate its possible identity,
bacterially expressed purified PKI
-78 was incubated with
cyclin-dependent kinase (Cdk1; gift of Dr. Richard Vulliet,
Department of Molecular Biosciences, University of California, Davis,
CA), given the presence of a Ser-Pro sequence within the PKI
protein. 32P-Labeled PKI
-78, as produced by such a
reaction, comigrates with the spot termed phospho-PKI
-78 on the
two-dimensional Western blot (Fig. 5, lower panels), thus
providing evidence that PKI
-78 likely also exists in phospho and
dephospho forms. The phosphorylation of either PKI
-70 or PKI
-78
by Cdk1 was only achieved to a modest degree (~2% stoichiometry) and
with a very high Km value (>100 µM).
It is thus quite unlikely that under physiological conditions, Cdk1
catalyzes this PKI
phosphorylation, but other Ser-Pro-directed
kinases, of which there are now many, might be the responsible in
vivo catalyst. The 32P-labeled PKI
-78 species
generated with Cdk1 and as used in the experiment presented in Fig. 5
is, however, likely to be a reliable marker to indicate the migration
of a species of PKI
-78 that contains 1 mol of phosphate (even though
the phosphate incorporated may not necessarily be in the same site as
in the native testis protein). A protein kinase capable of
phosphorylating PKI
-78 has been identified in male germ
cells.2 A 32P-labeled form of
bacterially expressed PKI
-70 was also obtained by incubation with
Cdk1, which likewise produced the characteristic change in isoelectric
focusing migration (data not shown).
Quantification of PKI
The relative distribution of the PKI isoform in
adult rat testis and cerebellum was determined from scans of the
two-dimensional Western blots of adult rat testis and cerebellum
extracts. A range of protein amounts (0.7, 1.2, 1.7, 2.3, and 3.5 µg)
was applied to the two-dimensional gels to validate that the signals
for each of the PKI
isoforms were in the linear range of response.
This allowed for an accurate determination of the relative percentage composition of the different forms. The results of quantification by
this procedure are presented in Fig. 4 (b and c).
As is evidenced, in testis, PKI
-70, PKI
-X, and PKI
-Y
constitute ~39, 23, and 32% of the total, respectively, each with a
very similar proportion (35-42%) of monophospho versus
dephospho forms. Rat cerebellum contains only the dephospho forms of
PKI
-70, PKI
-X, and PKI
-Y, with markedly less PKI
-70 than in
testis and with PKI
-Y in the highest abundance. PKI
-78 is present
only in low amounts in either testis or cerebellum (~2-6% of the
total) and almost entirely as the monophospho species. The difference
in the relative percentage of phosphorylated to nonphosphorylated
PKI
-78 in comparison with what is seen for the other PKI
species
may in some manner be related to the differences in sensitivity that
were observed with alkaline phosphatase treatment; the
phospho-PKI
-78 form was observed to be markedly more resistant to
hydrolysis (see Fig. 5). Such a difference might arise, as an example,
if the site of phosphorylation in PKI
-78 was different than in the
other phospho-PKI
forms.
The higher molecular mass PKI-X and PKI
-Y
species have been identified based upon their cross-reactivity with
both anti-PKI
-(5-22) and anti-PKI
-(60-70) antisera. Each
exhibits an antigenicity with the two antibodies very similar to that
obtained with both PKI
-70 and PKI
-78. The PKI
-(5-22) peptide,
against which the first of these two antibodies was prepared, contains
the pseudosubstrate PKA inhibitory site, and the peptide itself is
nearly as equipotent a PKA inhibitor as the full-length protein (5).
Given the observed antigenicity, it might be anticipated that PKI
-X
and PKI
-Y would exhibit PKA inhibitory activity. This was documented
by the further purification of PKI
-X and PKI
-Y by a combination
of DEAE chromatography, gel filtration, and affinity
chromatography.
Rat cerebellum contains high levels of both PKI and PKI
. These
are readily separated by DEAE chromatography (Fig.
6a), with first the elution of multiple forms
of PKI
(1-3-millisiemens conductivity), followed by the subsequent
elution of PKI
(5-6-millisiemens conductivity). The multiplicity of
PKI
forms was examined by Western blot analyses using both
anti-PKI
-(5-22) and anti-PKI
-(5-22) antibodies (Fig. 6,
b and c). No antigenic cross-reactivity between the PKI
and PKI
forms eluted from the DEAE column was detected (data not shown). Three overlapping peaks in the 1-3-millisiemens conductivity range of PKI
were observed for the elution of rat cerebellum protein from the DEAE column. The first (fractions 12-14)
contains predominantly PKI
-70, the second (fractions 16-18) contains PKI
-X plus the lower levels of PKI
-78, and the third (fractions 18-21) contains PKI
-Y. Fractions 21-23 also show the presence of a low abundance species of slightly higher apparent molecular mass than PKI
-Y. This is likely to be "PKI
-Z," a
form that is a more prominent constituent of epididymal sperm and is discussed further in the accompanying paper (17). PKI
is eluted from
the DEAE column at markedly higher conductivities (fractions 50-67)
and well separated from the PKI
forms (Fig. 6, a and
c). Multiple charge forms of PKI
were evident in DEAE
separation, as has been described previously for skeletal muscle and
brain PKI
(29, 30). The major forms of cerebellum PKI
comigrated with purified skeletal muscle PKI
(data not shown). A lower
molecular mass species that is evident in later eluting fractions on
the Western blots is likely identical to the skeletal muscle PKI
-I
form described previously (28, 29).
The DEAE elution of PKI forms from rat testis (Fig. 6d)
showed a similar pattern to that from rat cerebellum, made more
complicated, however, by the presence of the phospho forms of
PKI
-70, PKI
-X, and PKI
-Y. These latter species are extensively
present in testis, but absent in cerebellum (see Fig. 4). Three
prominent peaks of inhibitor activity are evident from the DEAE elution
of the rat testis extract. The first (fractions 14-16) contains almost
exclusively dephospho-PKI
-70 and was the fraction that had been
previously used in the first elucidation of the PKI
form of
inhibitor protein (5). The second peak (fractions 19-21) contains
PKI
-78 plus dephospho-PKI
-X. Fractions 23-27 contain
phospho-PKI
-70, phospho-PKI
-X, and dephospho-PKI
-Y.
Phospho-PKI
-Y is then eluted in the subsequent fractions, 30-34.
Identification of these different forms was confirmed by
two-dimensional analyses (data not shown).
Each of the two DEAE profiles of elution of PKI from cerebellum and
testis (Fig. 6, a and d) shows a strong
coincidence between PKA inhibitory activity and the elution of the
higher molecular mass PKI
forms. Further confirmation that both
PKI
-X and PKI
-Y are inhibitors of PKA was obtained by subsequent
gel filtration. Pools I (fractions 12-14), II (fractions 16-17), and
III (fractions 19-22), obtained by the DEAE chromatography of
cerebellum extracts (Fig. 6a), were each lyophilized and
then applied to a Sephacryl S-100 gel filtration column. This
purification fully separated the higher and lower molecular mass
species of PKI
(Fig. 7, a-d) and
demonstrated that both PKI
-X (pool II) and PKI
-Y (pool III) were
as effective inhibitors of PKA as the lower molecular mass forms.
Equivalent results were also obtained by gel filtration of the testis
PKI
fractions obtained by DEAE fractionation, again illustrating the
PKA inhibitory activity of PKI
-X and PKI
-Y (data not shown).
A further purification of both cerebellum PKI-X and PKI
-Y was
achieved using PKA catalytic subunit affinity chromatography, which, in
the case of PKI
-Y, yielded a protein that was at least 65% pure
(Fig. 8, inset a). The conditions used for
this affinity chromatography were identical to those described
previously (31), except for the use of the recombinant PKA catalytic
subunit. PKI
-Y, obtained by SDS gel electrophoresis, was in-gel
digested with protease Lys-C, the eluted peptides were then digested
with protease Glu-C, and peptides were separated using a Vydac
C18 narrow-bore reverse-phase HPLC column with a
trifluoroacetic acid/acetonitrile gradient. The major peptide obtained
from PKI
-Y in sufficient amounts for protein sequencing yielded the
sequence
SVISSFASSARAGRRNALPDIQSSLAT ... This 27-amino acid peptide is identical in sequence to residues 5-27
of PKI
-70. It contains the pseudosubstrate inhibitor domain, and the
recognition determinant residues are denoted in
boldface.3 This establishes that PKI
-Y
contains the identical pseudosubstrate region as identified in
PKI
-70 and also fully accounts for the antigenicity observed against
anti-PKI
-(5-22). Near equal, if not equal, antigenicity of
PKI
-70, PKI
-78, and PKI
-Y with anti-PKI
-(5-22) antibody
would be expected.
The affinity-purified preparations of PKI-X and PKI
-Y were tested
for protein kinase inhibitory activity. As is illustrated in Fig. 8,
both are efficacious inhibitors of PKA. Adequate amounts of protein
have not yet been available for a full analysis of inhibitory potency.
Estimates of protein based upon Coomassie Blue staining on SDS gels
(Fig. 8, inset a) and antigenicity suggest, however, that
PKI
-X and PKI
-Y have PKA inhibitory potencies very similar to
those of PKI
-70 and PKI
-78. The concentration of PKI
-X used in
the experiment depicted in Fig. 8 was ~18% of that of PKI
-Y,
accounting for the difference in the profile. PKI
-Y and PKI
-X
were also tested as inhibitors of the cGMP-dependent protein kinase. In total contrast to what has been observed with both
PKI
-70 and PKI
-78 (see Fig. 1) and also with PKI
(26), none of
which inhibit PKG even at very high concentrations, PKI
-Y and
PKI
-X were quite effective inhibitors of the
cGMP-dependent protein kinase (Fig. 8). Based upon the
IC50 values, the Ki values for PKG
inhibition by these two forms are in the 1-5 nM range. In
an earlier report by Szmigielski et al. (32), two forms of
protein kinase inhibitor were isolated from rat cerebellum, termed
by those authors types I and II. Type I was specific for PKA, whereas
type II inhibited both PKA and PKG. Based upon these inhibitor
potencies and their reported gel filtration elution profile (32), it is
now apparent that type I is PKI
and type II is a combination of
PKI
-Y and PKI
-X. This is fully consistent with the DEAE profile
of PKI distribution in cerebellum that is illustrated in Fig.
5a. The low level of PKI
-70 observed in this profile
would likely have not been detected in the separation applied by
Szmigielski et al. (32). Thus, the PKG inhibition now
observed for PKI
-Y and PKI
-X (Fig. 8) is consistent with the past
literature. The inhibition of PKG by PKI
-Y is of note given the
identified pseudosubstrate sequence that this inhibitor possesses.
Short PKI
peptides, unlike their parent protein, do inhibit PKG to a
modest degree (26, 33), and PKI
-based substrate peptides are quite
good substrates for this enzyme (34). Clearly, it is either the other
component parts of this PKI
-Y that must aid in enhancing its
interaction with PKG or some component part of PKI
-70 and PKI
prohibits PKG interaction. Either is of interest given that these two
protein kinases are closely homologous. Despite being less efficacious
with PKA than with PKG, PKI
-Y and PKI
-X may be effective
inhibitors of PKG physiologically since the cellular concentrations may
well exceed the association constant for the interaction.
The results presented here open up a new level of complexity in
the already intricate nature of the cAMP signal transduction pathway.
As has been well established, multiple forms of the protein constituents of PKA exist (35, 36). The three primary species of the
catalytic subunit (C, C
, and C
) are the products of separate
genes, and each has a distinctive cellular and tissue distribution
pattern. As yet, however, there is little evidence that these three
species in vivo catalyze the phosphorylation of distinct
proteins, except possibly some yet to be fully explored indications
that the C
isoform might exhibit some differences in substrate
preference (37). It is possible that the major reason for the existence
of multiple PKA catalytic forms is in order for their transcriptional
expression to be differentially controlled and, in doing so, to create
the different cellular milieu of PKA activity in each cell. Much,
however, remains to be determined about the physiological events of
PKA-dependent phosphorylation. With the presence of
multiple protein substrates in a single cell, there is a need for not
only each substrate to be phosphorylated in response to a cAMP signal,
but also for there to be some preference in the order of their
phosphorylation and by this means to orchestrate the integration of the
cellular response (38). Possibly the existence of multiple catalytic species contributes to such events. There are also multiple forms of
the PKA regulatory subunit. The two primary types, RI and
RII, differ in some aspects of their cAMP sensitivity and
in the existence in RII of cellular targeting sequences
(39, 40). Why there is a need for there to be
and
species of
each, however, remains to be identified (41). Possibly they too exist
to allow for differential transcriptional expression in different cell
types and/or to allow for the complexity of response to the cAMP signal that occurs with each cell. Multiple species of AKAP proteins have also
been identified that are involved in distributing the cAMP signal
throughout the cell and by doing so would allow for a
differential response dependent upon subcellular localization of
the kinase substrates (39, 42).
We have now demonstrated that PKI also exhibits a multiplicity of
species. Previously, we established that there are two gene products,
PKI and PKI
(6), and these current studies now show that PKI
exists in eight or more different forms as related by at least covalent
modification and alternate translational initiation. The first evidence
of multiple species of a testis-derived PKI in fact preceded these
current studies. PKI from testis was initially described by Means and
co-workers (12, 43), and as judged from its DEAE-cellulose elution
characteristics, the form isolated by them was likely one (or more) of
the isoforms of PKI
. Subsequently, these same investigators
generated antibodies against this purified testis PKI and, by their
use, identified four forms of the protein in testis extracts. The two
most prominent species had molecular masses of 9.3 and 15.6 kDa (27),
closely similar to what we now observe for PKI
. Our current data are fully complementary to the initial reports of Means and co-workers.
One mode for the formation of different PKI isoforms appears to be
by alternate translational initiation, giving rise to at least
PKI
-78 and PKI
-70. According to the Kozak scanning model of
translation, a ribosome binds to the 5
-end of the mRNA and
proceeds downstream until an AUG codon is encountered (13). Most often,
translation is initiated at the first AUG codon, although the
efficiency of translation is dependent upon the sequence context of
this AUG codon. Where there are two or more in-frame AUG codons within
a short distance of each other (<20 codons), alternate translational
initiation at the second AUG codon has also been observed. The
synthesis of multiple forms of brain creatine kinase (44) and the yeast
protein MOD5 (45) are two examples where translation is initiated at
both the first and second AUG codons within a short stretch of
mRNA. It appears most likely that PKI
-70 and PKI
-78 arise by
such an alternate initiation of translation. Both of the AUG codons in
PKI
, which would give rise to these two forms of the protein, have
initiation sequences that would readily allow translation to be
initiated at either site (13). Neither, however, has the optimum Kozak
sequence, and so read-through of the first upstream AUG codon to allow
initiation at the second is thus likely. Although it remains a
possibility that PKI
-70 could arise instead as a consequence of the
proteolytic cleavage of PKI
-78, this seems unlikely. The
amino-terminal sequence of PKI
-70 is consistent with its being
directly synthesized and then processed to remove the terminal
methionine and to add a blocking group (14-16). In both testis and
cerebellum, PKI
-70 (as both phospho and dephospho forms) is much
more prevalent than PKI
-78 (Fig. 4, b and c).
This may be a consequence of the second AUG codon being the preferred
initiation site or because PKI
-78 has a shorter half-life. The
translation efficiency of a given AUG codon is affected by many
parameters, including the sequence context of the AUG codon, secondary
structure of the mRNA, and length of the 5
-untranslated region
(13). PKI
-70 and PKI
-78 have equivalent PKA inhibitory activity.
As one possibility, the production of these two forms of PKI
may be
important to direct them to different intercellular locations, as
occurs with the multiple forms of MOD5 protein that are produced by
alternate translational initiation (45). The additional 8 amino-terminal amino acids of PKI
-78 do not, however, conform to any
established signal sequences.
The second mode for the formation of alternate PKI isoforms is by
their post-translational phosphorylation, which gives rise to the
phospho forms of the four species. The degree of change in isoelectric
point between the phospho and dephospho forms of each suggests that
each is likely to be monophosphorylated. We have yet to determine which
enzyme(s) catalyzes the phosphorylation of the PKI
isoforms. We have
shown previously that PKI
could be phosphorylated by the epidermal
growth factor tyrosine kinase receptor (47); however, since neither
PKI
-70 nor PKI
-78 contains tyrosine residues, an equivalent
phosphorylation cannot explain the formation of phospho forms observed
with at least these two lower molecular mass species of PKI
. The
differential sensitivity of phospho-PKI
-70 and phospho-PKI
-78 to
protein phosphatase treatment suggests that possibly more than one
kinase may be involved, and the fact that the phospho forms are more
predominant in testis than in cerebellum may indicate that the protein
kinase responsible exhibits a distinctive tissue distribution pattern.
One example of such a kinase is the male germ cell-specific
Ser-Pro-directed kinase (Mak) that appears to be present only in
post-meiotic germ cells in testis (48, 49). As of yet, there is no
evidence that phosphorylation changes the inhibitory potential of the
PKI
forms and, as expanded upon below, is probably unlikely as a
physiological mechanism of control.
The molecular identity of PKI-X and PKI
-Y remains to be
elucidated. Neither PKI
-X nor PKI
-Y cross-reacts with the
PKI
-(5-22) antisera. Both showed equivalent reactivity to both the
anti-PKI
-(5-22) and anti-PKI
-(60-70) antisera, suggesting
substantial sequence homology to both PKI
-70 and PKI
-78, and
PKI
-Y contains at least residues 5-27 of PKI
-70. PKI
-X and
PKI
-Y appear unlikely to be the "b-2" species suggested to arise
as a consequence of alternate RNA splicing in mouse (8) since neither
comigrated with that form when bacterially expressed. The observation
that both PKI
-X and PKI
-Y can also inhibit PKG (Fig. 8) is
compatible with the earlier observations of Szmigielski et
al. (32) and suggests a key difference in their molecular
structure in comparison with PKI
-70 and PKI
-78. The structure of
these higher molecular mass forms is under current evaluation.
The specific functions of the eight forms of PKI remain to be
elucidated. While possible, it would appear unlikely that these represent PKI forms with different levels of PKA inhibitory potency. The concentration of PKA and PKI in cells exceeds the
Ki value of inhibition by several orders of
magnitude, and thus, even a very large change in the
Ki value between forms may not result in a greater
or lesser degree of PKA activity under physiological conditions. It
appears more probable that the different forms of PKI
would play
some role in modulating the full cAMP response, other than the simple
direct block of PKA activity, and be involved in such activities as
modulating the intracellular site of the PKA catalytic subunit (2-4,
9) or allowing for an altered pattern of interaction with its
regulatory subunit.
The preparation of the anti-peptide
antibodies, the procedure for Western blot analysis, and the studies of
multiple forms of PKI by one-dimensional electrophoresis were
accomplished by David Harrison, who also participated in some of the
two-dimensional gel characterization.