From the
Collagen IV is the major component of basement membranes. The
human
Goodpasture syndrome, an autoimmune disorder that has been
described only in humans, is mediated by circulating antibodies against
the carboxyl-terminal region of the human
Recent studies have
reported both the exon/intron and derived primary structures of the
Goodpasture antigen (Morrison et al., 1991; Turner et
al., 1992; Quinones et al., 1992) and have established
that the gene region encompassing the human antigen undergoes
alternative splicing, a phenomenon uncharacteristic of
Recombinant
antigens (22 µg/ml) were similarly phosphorylated with 9 units of
PKA catalytic subunit (Promega) and 10 µCi of
[
To perform renaturation assays of protein kinases on Western
blots, purified PKA catalytic subunit and plasma membrane were analyzed
in SDS-PAGE on 10% gels. After electrophoresis, the proteins were
transferred to Immobilon P (Millipore) in the absence of methanol. The
membrane was washed five times with 10 ml of 40 mM Hepes, pH
7.4, during 10 min. Fragments of membrane containing the protein
kinases of interest were excised and independently incubated at room
temperature with 100 µl of the corresponding protein substrate (50
µg/ml in Hepes buffer). After 10 min, 100 µl of the same buffer
containing 20 mM MnCl
When the phosphorylation of GPpep1 by the PKA
holoenzyme was assayed, a total dependence on cAMP was observed. The
presence in the assay of the protein inhibitor specific for this kinase
inhibited 80% of its activity on GPpep1, a similar value to that
obtained with histone type IIA (data not shown). The apparent
K
To further characterize the phosphorylation of
GPpep1 by PKA, the reaction mixture was applied to an HPLC
µBondapack
These
data strongly suggest that the Goodpasture antigen is phosphorylatable
at Ser
However,
the phosphorylation of GPpep1 does not imply that similar
phosphorylation could occur in the full antigen. To test this, we
produced in a bacterial expression system recombinant Goodpasture
antigen (rGP) and derived mutants in which one or both of the two
serines at the amino side conforming to a PKA phosphorylation consensus
site (GLKGKRGDS
Because the native
structure of the Goodpasture antigen is maintained by cystines and the
recombinant material was produced in the reducing environment of
bacteria, the rGP did not display native folding (Penadés et
al., 1995). Therefore, it was of interest to investigate if the
native antigen would also be similarly phosphorylated. In
Fig. 3
we show that the native Goodpasture antigen is
phosphorylated by the catalytic subunit of PKA (A, lane 1) and
that its amino-terminal region contains major phosphorylation sites
(B, lane 1), resembling the phosphorylation of the recombinant
antigen. However the nonavailability of mutants from natural source
with specific serine substitution makes it impossible to determine the
contribution of each individual serine to the phosphorylation event.
Nevertheless, because 1) the bovine and human antigens have a 90%
sequence identity with divergencies concentrated at the amino-terminal
region, including Ser
From these findings we conclude that
the human kidney plasma membrane contains a 50-kDa protein kinase
activity capable of phosphorylating rGP and the existence of a plasma
membrane-bound PKA is suspected.
To further investigate the nature
of these protein kinases, we renatured membranes after Western blot
analysis. The individual protein kinases were excised, and their
capability to independently phosphorylate different substrates tested.
In A of Fig. 7we show the phosphorylation of rGP
(lane 1), -26rGP (lane 2), and GPpep1 (lane
3) by the 50-kDa protein kinase. Similar results were obtained
with the bovine catalytic subunit of PKA and with the 41-kDa protein
kinase from the plasma membrane sample (data not shown). The
phosphorylation of GPpep1 and the reduction in
The data presented in this report strongly suggest that the
unique five-residue motif at the amino-terminal end of the Goodpasture
antigen, also present in all but one of its truncated alternative
forms, is a major in vivo serine phosphorylation site. In
recent reports, we proposed that this motif may be involved in cell
attachment to
Cell surface-associated cAMP-dependent and cAMP-independent
ecto-protein kinases have been found in a number of different cell
types, including human fibroblast derived cell lines (Kubler et
al., 1982, 1989). In this report, we present evidence supporting
the existence of at least two type A protein kinases associated with
human kidney plasma membranes that can specifically phosphorylate
KRGDS
It is premature to confirm if the association between plasma
membrane and PKA is due to cytosolic PKA trapped in the sample
preparation or is indeed PKA bound to the plasma membrane. In the more
likely latter case, it would be of great relevance to determine if the
membrane bound pool of this protein kinase is functionally linked to
the cytosolic pool as occurs for other protein kinases (Azzi et
al., 1992). If so, the more likely location would be the cytosolic
side of the plasma membrane, and therefore, it would not be a good
candidate to perform physiological phosphorylation of an extracellular
protein like the Goodpasture antigen. It has been shown that PKA type I
distributes randomly in the cytosol of resting T-cells while localizing
at the plasma membrane inner surface after receptor activation
(Skalhegg et al., 1994). On the contrary, the 50-kDa protein
kinase was not detected in cytosol and appears to be a plasma membrane
protein and, therefore, a plausible candidate to carry out the in
vivo phosphorylation of the Goodpasture antigen. It is remarkable,
however, that the Goodpasture antigen is a substrate for PKA.
Therefore, one could imagine that pathological release of PKA could
have profound effects on the phosphorylation steady state of the
antigen, thereby affecting immunoreactivity. Phosphorylation-dependent
epitopes have been shown to be important to the onset of another
autoimmune disorder, experimental systemic lupus erythematosus (Stetler
et al., 1992). During the initial stages of this disease, the
majority of the autoantibodies are directed against phosphorylated
epitopes. As the disease progresses, however, there is an increase in
the proportion of autoantibodies against phosphorylation-independent
epitopes which become the major ones in the established disease.
Immunoblot studies using Goodpasture serum and the two Goodpasture
fractions, GPs and GPp, did not show different immunoreactivities (data
not shown). However, if Goodpasture syndrome progresses in the same
fashion, clinical diagnosis of patients with established autoimmune
response could preclude the detection of autoantibodies against
phosphorylation-dependent epitopes. Alternatively, it may be necessary
to perform more quantitative studies using more adequate material
(non-phosphorylated natively folded recombinant material and highly
in vivo phosphorylated Goodpasture antigen fractions) to
detect residual phosphorylation-dependent immunoreactivity. In addition
to the Goodpasture antigen, in vivo phosphorylation and the
capacity to be phosphorylated by PKA in vitro are features
common to at least two other autoantigens, acetylcholine receptor and
myelin basic protein (Ferrer-Montiel, et al., 1991; Kishimoto,
et al., 1985). All this suggests that phosphorylation may
serve as a common link in the onset of, at least, some forms of
autoimmunity.
GPpep1 and an unphosphorylatable control
peptide, GPpep2, were phosphorylated with different protein kinases. At
the indicated times, aliquots (25 µl) were taken and assayed for
We express our gratitude to Javier Alcácer,
Jerónimo Forteza, Samuel Navarro, and Joaqun Ortega for
providing human kidney samples. The technical assistance of Mara
José Agulló and Mara Francisca Ripoll is greatly
appreciated.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
3 chain of collagen IV contains an antigenic domain called
the Goodpasture antigen that is the target for the circulating
immunopathogenic antibodies present in patients with Goodpasture
syndrome. Characteristically, the gene region encoding the Goodpasture
antigen generates multiple alternative products that retain the antigen
amino-terminal region with a five-residue motif (KRGDS). The serine
therein appears to be the major in vitro cAMP-dependent
protein kinase phosphorylation site in the isolated antigen and can be
phosphorylated in vitro by two protein kinases of
approximately 50 and 41 kDa associated with human kidney plasma
membrane, suggesting that it can also be phosphorylated in
vivo. Consistent with this, the Goodpasture antigen is isolated
from human kidney in phosphorylated and non-phosphorylated forms and
only the non-phosphorylated form is susceptible to phosphorylation
in vitro. Since this motif is exclusive to the human
3(IV) chain and includes the RGD cell adhesion motif, its
phosphorylation might play a role in pathogenesis and influence cell
attachment to basement membrane.
3 chain of collagen IV
(
3(IV)), also called the Goodpasture antigen. The antigen consists
of a short collagenous Gly-X-Y sequence followed by
the NC1
(
)
domain (Saus et al., 1988).
Since
3(IV) is also present in other species, and at least six
related chains (
1-
6) exist in human collagen IV, a
fundamental question is whether peculiarities in the molecular
structure and/or in the biology of the human antigen are important for
the characterization of the autoimmune response.
3(IV)
chains from other species and not described for other human collagen IV
chains (Bernal et al., 1993; Feng et al., 1994;
Penadés et al., 1995). All the alternative mRNAs
isolated encode truncated forms expected to be nonfunctional in triple
helix formation and all but one retain the amino-terminal region of the
Goodpasture antigen, previously identified as the most divergent
sequence known among the carboxyl-terminal domains of collagen IV
(Quinones et al., 1992). A unique feature of this
amino-terminal sequence when compared with other collagen IV chains is
the overlapping positioning in a five-residue motif of consensus sites
for cell adhesion and for the action of a number of protein kinases
(KRGDS
) (Quinones et al., 1992). The data
presented in this report demonstrate that cAMPdependent protein kinase
(PKA) phosphorylates this motif in vitro and strongly suggest
that it comprises the major in vivo serine phosphorylation
site of the Goodpasture antigen. Also we identify two protein kinases
associated with the plasma membrane that can phosphorylate the human
antigen at this specific serine, and we discuss the possible role and
implications of these findings with respect to the cell attachment to
basement membrane and to the autoimmune disorder.
Materials
Fragments of histologically normal human renal cortex from
two nephrectomies and human kidneys from necropsies were stored at
-70 °C until use in plasma membrane and Goodpasture antigen
preparations, respectively. PKA holoenzyme, its catalytic subunit, and
casein kinases 1 and 2 were purified from rat liver cytosol according
to Guasch et al.(1986). When indicated, the catalytic subunit
of PKA used was from Promega. Protein Kinase C was purified from rat
brain as indicated in Walker and Sando(1988).
[-
P]ATP was from Amersham Corp. All other
materials were of analytical grade and were purchased from Sigma or
Boehringer Mannheim.
Synthetic Polymers
Oligonucleotides
The sequence of each of the
oligonucleotides (Biotech, National Biosciences, Oligos Etc.,
Pharmacia) used is given in the 5`- to 3`-end direction. The
corresponding encoded protein sequence is indicated in one letter code.
The base or amino acid representing changes with respect to the native
sequences are underlined. The oligonucleotides used were ON-BHNC1c
(antisense), CAG-BamHI site-GTTCTTTAGGATGAAAA (3`-untranslated
region); ON-HNC5m (sense), CAG-BamHI site-AGGTTTGAAAGGAAA
(GLKGK); ON-HNC9m (sense), CAG-BamHI site-TCAAACCACAGCAATTC
(QTTAI); ONGP-Ser (sense), CAG-BamHI
site-AGGTTTGAAAGGAAAACGTGGAGACGCTGGATCACCTGC (GLKGKRGDAGSPA);
ONGP-Ser1c (antisense), GTGGTTTGAGCGTGTCGGGT (TRHAQTT); ONGP-Ser2m
(sense), ACCCGACACGCTCAAACCAC (TRHAQTT).
Peptides
Peptides were synthesized by Multiple
Peptide Systems and are designated GPpep1
(KGKRGDSGSPATWTTRGFVFT-NH, consisting of residues
3-23 of the Goodpasture antigen),
(
)
GPpep3
(KGKRGDSG, consisting of residues 3-10 of the Goodpasture
antigen), and GPpep2 (MKKRH, the carboxyl-terminal sequence of the
Goodpasture antigen).
Antibodies
Monoclonal and polyclonal antibodies against GPpep1 were
produced following standard procedures. The aM2/M2 and anti-GPpep1/bov
antibodies were provided by Billy G. Hudson. The aM2/M2 antibodies
consist of rabbit antibodies against the 3(IV)NC1 (Saus et
al., 1988) that were affinity-purified on a Sepharose 4B matrix
containing covalently bound
3(IV)NC1. The anti-GPpep1/bov are
rabbit polyclonal antibodies raised against a synthetic peptide that
essentially represents the GPpep1 region of the bovine antigen (Gunwar
et al., 1990). The affinity-purified antibodies against the
catalytic subunit of the PKA were obtained from an specific rabbit
anti-serum provided by Suzanne Lohmann. Monoclonal antibodies against
glyceraldehyde-3-P dehydrogenase and low density lipoprotein receptor
were provided by Erwin Knecht. Polyclonal antibodies against
Na
/K
-ATPase were provided by
Ramón Serrano. The monoclonal antibodies against phosphoserine
(PSer) were from Sigma. The conjugates used were anti-mouse IgG (H
+ L) and anti-rabbit IgG (Fc) alkaline phosphatase (Promega).
PCR Amplifications
PCRs were performed using as a template the previously
characterized cDNA (Quinones et al., 1992). Unless otherwise
indicated, 25 pmol of each primer were used in a 40-cycle amplification
program (94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1
min) in a total volume of 50 µl.
Plasmid Constructions and Purification of Recombinant
Proteins
Protein-expressing plasmid constructs were prepared by
digestion of the individual PCR amplification products with
BamHI and subsequent cloning into pET-22b vector (Novagen).
The oligonucleotides used in the PCR amplifications were ON-BHNC1c and
either 1) ON-HNC5 m, to produce rGP; 2) ON-HNC9 m, to produce a deleted
antigen lacking the first 26 amino acids (-26rGP); or 3)
ONGP-Ser, to produce a mutant substituting Ala for Ser (rGPAla9). To produce the other alanine-for-serine mutants
(rGPAla26 and rGPAla9,26), two separate PCR products with overlapping
sequence were combined following the protocol described in
Higuchi(1990). The oligonucleotide pairs used were ONGP-Ser2 m/ONBNC1c
and ONHNC5 m/ONGP-Ser1c (rGPAla26) or ONGP-Ser/ONGP-Ser1c (rGPAla9,26).
Initial clonings were established in Escherichia coli HB101,
and the cDNA of each plasmid of interest was further confirmed by
nucleotide sequence analysis. For protein expression, the plasmids were
transferred to E. coli BL21(DE3) cells, and the individual
clones were induced following the supplier's instructions. After
disrupting the cells by sonication, recombinant proteins were purified
by repeated precipitations.
Preparation of Goodpasture Antigen (
The 3(IV) NC1
Monomers) from Human and Bovine Kidneys
3(IV)NC1 monomers were prepared from NC1 of collagen
IV as in Hellmark et al.(1994). The HPLC fractions containing
only
3(IV)NC1 material were pooled and dried. This material was
resuspended in 50 mM Tris, 150 mM NaCl, pH 7.5 (GPs
fraction). The unsolubilized material was recovered with 8 M
urea in 50 mM Tris, pH 7.5 (GPp fraction). Unless indicated,
phosphorylation studies were done with GPp fractions by diluting the
antigen sample at least 100 times in phosphorylation buffer.
Phosphorylation Assays
Unless indicated, synthetic peptides (1 mg/ml) were incubated
at 30 °C as described (Guasch et al., 1986; Walker and
Sando, 1988) with [-
P]ATP and 0.5-1
unit/ml of the various kinases in the presence of 125 µM
ATP and 8 mM MgCl
. The reactions were terminated
by spotting an aliquot on Whatman P81 paper followed by washing in 75
mM H
PO
and assay of the radioactivity.
K
values were estimated using the
Enzfitter adjusting program (Leatherbarrow, 1987).
-
P]ATP during 15 min in a total volume of
30 µl. Native antigens (15-25 ng) were phosphorylated in 40
mM Tris, pH 7.5, 20 mM MgCl
, 150
µM ATP, 20 µCi of [
-
P]ATP,
and 25 units of PKA catalytic subunit (Promega) during 8-16 h in
a total volume of 50 µl.
HPLC Analysis of GPpep1 Phosphorylation
GPpep1 was incubated with [-
P]ATP
and the PKA catalytic subunit. After 6 h, the reactions were terminated
by the addition of trifluoroacetic acid to a concentration of 0.5% and
injected into a µBondapack
C18 HPLC column (3.9
300
mm from Waters). The bound material was eluted with a 30-min gradient
of 0-60% acetonitrile in 0.05% trifluoroacetic acid at a flow
rate of 1 ml/min. The absorbance of the effluent was monitored
continuously at 214 nm, 1-min fractions were collected, and Cerenkov
radiation was determined. Both phosphorylated and unphosphorylated
GPpep1 eluted as a single peak at 22 min.
Phosphoamino Acid Analysis
The HPLC fractions containing the phosphorylated peptide were
combined, dried, dissolved in 6 M HCl, and incubated 1 h at
110 °C. The HCl was then removed by evaporation in a rotavapor and
the residue was redissolved in water. After addition of carrier
unlabeled phosphoamino acids, the solution was subjected to
electrophoresis (1000 V for 1 h) in cellulose thin layer plates using a
buffer of pyridine:acetic acid:water, 5:50:945, pH 3.5. Amino acids
were revealed with ninhydrin, and the radioactivity was detected
autoradiographically.
Isolation of Plasma Membrane
Human kidney plasma membranes were prepared essentially as
described in Von der Mark and Risse(1987). After saccharose gradient,
the interphase 17-40% containing the plasma membranes were
collected and stored at -20 °C until needed. Where indicated,
partially purified membrane fractions and soluble material were used
for renaturation assays.
Renaturation Studies
Renaturation and assay of protein kinases after SDS-PAGE was
performed essentially as in Hutchcroft et al.(1991) using 10%
gels.
and 20 µCi of
[
-
P]ATP was added, and the mixture was
incubated for 3 h 30 °C in a water bath. Mn
has
been shown to be much more effective than
Mg
-supporting PKA catalytic subunit action in
renaturation studies at low ATP concentrations (Geahlen et
al., 1986). Subsequently, soluble material was analyzed by
SDS-PAGE prior or after digestion and immunoprecipitation as indicated
below. When assaying GPpep1, the individual membranes were blocked (3%
bovine serum albumin in Hepes buffer for 10 min) and extensively washed
(Hepes buffer) prior to the addition of 200 µl of phosphorylation
mixture containing 20 nmol of GPpep1.
Immunoprecipitation of V8 Endoproteinase-digested
Goodpasture Antigens
When indicated, the phosphorylation mixture was reduced and
digested in 50 mM NH acetate, pH 4, 10 mM
dithiothreitol using 0.5 µg of V8 proteinase. After 14 h at 37
°C, the mixture was neutralized with 1 M Tris, pH 8.8, 10
mM dithiothreitol was added to maintain reducing conditions,
and the digested material was alkylated for 1 h on ice with 20
mMN-ethylmaleimide. The mixture was brought to
oxidizing conditions with 1 µl of 33% H
O
and heated 15 min at 75 °C. This material was diluted
10-20 times with 10 mM Tris-HCl, 150 mM NaCl,
0.05% Tween 20, pH 8, and incubated with 50 µl of pre-immunized
control serum for 1 h at room temperature. The control antibodies were
removed using protein A-Sepharose. Subsequently, the precleared
supernatant was similarly extracted with the same amount of specific
antisera (anti-GPpep1 or anti-GPpep1/bov). The protein A precipitates
containing the materials of interest were analyzed by SDS-PAGE and
autoradiography.
Nucleotide Sequence Analysis
All the constructs to be expressed were sequenced by the
dideoxy chain termination method (Sanger et al., 1977) using
S-dATP (Biggin et al., 1983), modified T7 DNA
polymerase (Tabor and Richardson, 1987), and universal and
3(IV)-specific primers.
Physical Methods and Immunochemical Techniques
SDS-PAGE, protein transference onto Immobilon P membrane
(Millipore), and immunochemical techniques (enzyme-linked immunosorbent
assay and Western blotting) were performed essentially as described
(Laemmli, 1970; Burnette, 1981; Johansson et al., 1992).
Other Methods
Where indicated NH-terminal sequence was
determined using an Applied Biosystems gas-phase protein sequencer
model 470A by the Servei de Sequenciació, Universitat de
Barcelona.
In Vitro Phosphorylation of the Amino-terminal Region
of the Goodpasture Antigen by PKA
A 21-mer peptide, GPpep1
(KGKRGDSGSPATWTTRGFVFT), representing residues 3-23 of the
Goodpasture antigen was assayed for its potential to be
phosphorylated. Among the several protein kinases tested, only the
catalytic subunit of PKA significantly phosphorylated this peptide
().
(Michaelis-Menten constant) value for
GPpep1 phosphorylation was 0.68 mM, a value similar to the
K
values found for other peptide
substrates of PKA (Kemp et al., 1977; Zetterqvist and
Ragnarsson, 1982).
C18 column and eluted as indicated under
``Experimental Procedures.'' As shown in
Fig. 1A, the major absorbance and radioactive peaks were
coincident and eluted at 22 min. Fractions corresponding to this peak
were used then for phosphoamino acid and amino-terminal sequence
analysis (Fig. 1, B and C, respectively). From
these data we conclude that GPpep1 is phosphorylatable by PKA at serine
residues.
Figure 1:
HPLC analysis of the GPpep1 incubated
with catalytic subunit of PKA in the presence of
[-
P]ATP. The figure represents the
absorbance at 214 nm (- - - -) and the
radioactive (--) profiles obtained when a sample of GPpep1
incubated with PKA catalytic subunit in the presence of
[
-
P]ATP was applied to a
µBondapack
C18 HPLC column and eluted with the gradient
indicated under ``Experimental Procedures'' (A).
Phosphoamino acid and amino acid sequence analyses of the fractions
corresponding to the peak eluting at gradient times 22 and 23 min are
shown in B and C, respectively. In B,
arrows indicate the migration position of the indicated
phosphoamino acids standards and the starting point (O) in the
chromatogram.
Of the two serines in GPpep1,
KGKRGDSGS
PATWTTRGFVFT, that at position 9
within the Goodpasture antigen conforming to the consensus motif for
PKA recognition (K-R-X-X-S) (Kemp and Pearson, 1990)
appears to be the most likely point of phosphorylation. In fact, GPpep3
(KGKRGDS
G) a shorter peptide containing only this serine
was also phosphorylatable, and a GPpep1 variant in which Ser
was replaced by Ala did not incorporated significant amount of
P in the same assay conditions (data not shown).
by PKA in the region where maximum differences exist
between the different NC1 domains of collagen IV chains.
GSPATWTTRGFVFTRHS
) are replaced
by alanines (rGP-Ala9, rGP-Ala26, and rGP-Ala9,26) or the 26
amino-terminal residues containing both serines are removed
(-26rGP). In A of Fig. 2, we present the
autoradiographic analysis of the PKA-phosphorylated recombinant
proteins. The phosphorylation observed in rGP (lane 1) was
dramatically decreased in the mutants where both serines were removed
(lane 2) or replaced (lane 5). As expected, the
substitution of either serine resulted in reduced
P
incorporation (lanes 3 and 4). However, the
substitution of Ser
(lane 4) inhibited the
incorporation of
P to the recombinant antigen to a greater
extent, indicating that this is the preferred phosphorylation site in
the recombinant antigen. To further assess the region involved in the
P incorporation, we isolated and similarly analyzed the
corresponding amino-terminal regions of the phosphorylated proteins. To
achieve this, we specifically immunoprecipitated these regions after
digestion with V8 proteinase that specifically cuts the antigen after
Glu
. The autoradiographic patterns obtained (B)
were consistent with those of the full antigens, indicating that both
Ser are phosphorylatable by PKA and that they are the major
phosphorylation sites in rGP. The residual
P incorporation
in rGP-Ala9,26 and in -26rGP (A, lanes 2 and 5)
could be attributed to an additional PKA phosphorylation site located
more toward the carboxyl end (Ser
) and, therefore, not
present in the immunoprecipitated material (B, lanes 2 and
5).
Figure 2:
In vitro phosphorylation of rGP
and derived mutants by PKA catalytic subunit. One-tenth of the
corresponding phosphorylation mixtures was analyzed by reducing
SDS-PAGE (12.5%) and autoradiographed (A). The remaining
mixtures were reduced, digested, alkylated, immunoprecipitated, and
analyzed as in A in a 15% gel. The recombinant proteins are
rGP, -26rGP, rGPAla9, rGPAla26 and rGPAla9,26 (lanes
1-5, respectively). Arrowheads indicate the positions, from
top to bottom, of rGP and -26rGP (panel A) and the
specifically precipitated V8 digestion products of the recombinant
antigens (panel B).
From these data we conclude that the bacterial
recombinant form of the Goodpasture antigen is phosphorylatable by PKA
and that Ser and Ser
in its amino-terminal
region are the major phosphorylation sites.
(Thr in bovine sequence); and 2)
Ser
, Ser
, and the corresponding flanking
regions remain conserved (Turner et al., 1992; Quinones et
al., 1992), bovine antigen appears to be an excellent
``native human mutant'' where Ser
has been
substituted by Thr. Therefore, we carried out phosphorylation studies
using bovine antigen in order to assess the role of Ser
in
the in vitro phosphorylation of the native antigen structure
by the catalytic subunit of PKA (Fig. 3). Interestingly, we could
not detect incorporation of
P in the bovine antigen
(A, lane 2) nor in its amino-terminal region (B, lane
2) in the same assay conditions in which human antigen displayed
notable phosphorylation (A and B, lane 1). The
absence of
P incorporation in the bovine antigen suggests
that the sequences conforming the consensus sites therein (Ser
and Ser
) are either not easily accessible in the
native structure or that they are fully phosphorylated in
vivo. However, the latter explanation appears to be unlikely
because no phosphoserine is detectable in the isolated bovine antigen
(see below).
Figure 3:
In vitro phosphorylation of the
Goodpasture antigen from different sources by the PKA catalytic
subunit. Phosphorylation mixtures of human (lanes 1) or bovine
(lanes 2) native antigens were reduced and analyzed by
SDS-PAGE (8-18% gradient, A) or reduced, digested,
alkylated, immunoprecipitated with polyclonal antibodies against the
amino-terminal region of each antigen (anti-GPpep1 or anti-GPpep1/bov),
and then similarly analyzed by SDS-PAGE (B). The
phosphorylated material was visualized by autoradiography.
Arrowheads indicate the positions of antigens (A) and
their specifically precipitated proteolytic products (B). The
higher molecular weight autoradiographic bands in lanes 1 and
2 of A come from autophosphorylation of the PKA
catalytic subunit present in the assay.
From these comparative studies, it is firmly
established that the isolated native Goodpasture antigen is
phosphorylatable in vitro by PKA within its amino-terminal
region, and it is strongly suggested that Ser is the
preferred phosphorylation site.
The Goodpasture Antigen Is Phosphorylated at
Serines
To approach the physiological relevance of the
phosphorylation of the Goodpasture antigen, we investigated the content
of phosphoserine of native Goodpasture antigen in comparison with the
recombinant and bovine counterparts. Similar amounts of native (GP) or
recombinant antigen (rGP) were analyzed and blotted with monoclonal
antibodies against the amino-terminal region of the Goodpasture antigen
(M3/1) or with monoclonal antibodies against phosphoserine (PSer)
(Fig. 4A). A significant content of phosphoserine was
detected in the native antigen, whereas no reactivity was observed in
the bacterial recombinant form under the same assay conditions. The
PSer reactivity of native antigen was partially inhibited when
phosphoserine was added to the blotting antibodies but not when the
phosphoamino acid added was phosphotyrosine (data not shown). From
these data we conclude that the Goodpasture antigen is phosphorylated
in vivo at serines.
Figure 4:
Immunodetection of phosphoserine in the
Goodpasture antigen from different sources. Approximately 60 ng of
native (GP) or recombinant (rGP) human antigen
(A) and 30 ng of human (GP) or bovine (GPb)
native antigen (B) were analyzed by reducing SDS-PAGE
(8-18% gradient) and transferred to Immobilon P. The membranes
were split into two pieces following the longitudinal axis of each
sample well. Each individual half was blotted with monoclonal
anti-phosphoserine (PSer) and either M3/1 monoclonal
anti-GPpep1 (A) or affinity-purified polyclonal aM2/M2
(B). The position of the antigens are indicated by
arrowheads.
Because the presence or absence of
Ser appears to be a major contributor to the difference
between human and bovine antigens in their in vitro phosphorylation (Fig. 3), it was of interest to investigate
if differences could also be found in endogenous phosphoserine content.
In B of Fig. 4, similar amounts of human (GP) or bovine
(GPb) antigens were analyzed and blotted with polyclonal antibodies
that similarly react with both antigens (aM2/M2) or with
anti-phosphoserine monoclonal antibodies (PSer). Interestingly, bovine
material displayed no notable PSer reactivity in comparison with that
detected in the human antigen sample. The similarity between the in
vitro phosphorylation (Fig. 3) and the phosphoserine
contents (Fig. 4) of the native antigens suggests that Ser
could be the major in vivo serine phosphorylation site
in the Goodpasture antigen. If this is the case then the in vitro and in vivo phosphorylation of human antigen must be
inversely related, because only serines not phosphorylated in vivo can incorporate
P in the in vitro assays. We
have separated two human antigen fractions based on their distinct
ability to go into aqueous solutions after HPLC purification (see
``Experimental Procedures''). The antigen fraction that was
soluble in nondenaturing buffers (GPs) showed higher content in
phosphoserine when compared with the fraction that required 8
M urea to go into solution (GPp; Fig. 5A). When
these fractions were separately phosphorylated by the catalytic subunit
of PKA, the in vivo highly phosphorylated antigen (GPs)
displayed less ability to incorporate
P than the GPp
fraction (Fig. 5B). In one sample preparation, the GPp
fraction obtained did not contain any detectable phosphoserine and was
a much better substrate for in vitro phosphorylation studies.
In this case, the GPs fraction displayed more elevated PSer reactivity
and did not incorporate detectable
P in presence of PKA
catalytic subunit (data not shown).
Figure 5:
Phosphorylation of Goodpasture antigen
fractions by the PKA catalytic subunit. Approximately 15 ng of the
indicated Goodpasture antigen fractions were phosphorylated, analyzed
by reducing SDS-PAGE (8-18% gradient), and transferred to
Immobilon P. The membrane was autoradiographed at -70 °C for
several hours (B) and used for immunoblot detection. After
blocking the membrane was split and blotted as in A of Fig. 4
(A). Arrowheads indicate the positions, from top to bottom, of the PKA catalytic subunit and the
Goodpasture antigen.
All these data indicate that
Goodpasture antigen can be isolated in phosphorylated and
non-phosphorylated forms and strongly suggest that Ser is
the major in vivo phosphorylation site in the Goodpasture
antigen and that PKA or a catalytically related protein kinase is the
in vivo phosphorylating enzyme.
Identification of Two PKA-like Activities Associated with
the Plasma Membrane Which Can Phosphorylate the Amino-terminal Region
of the Goodpasture Antigen
To further study the significance of
the above phenomenon, we isolated plasma membrane from human kidney and
assayed renatured protein kinases after SDS-PAGE or Western blot
analysis. In the upper panels of Fig. 6, the catalytic
subunit of bovine PKA (lanes 1) or plasma membrane from human
kidney (lanes 2) were analyzed in SDS-polyacrylamide gels
polymerized in the absence or presence of different PKA protein
substrates. After electrophoresis and renaturation, the presence of
in situ phosphorylation, as a consequence of protein kinase
renaturation, was detected autoradiographically. In the absence of
protein kinase substrate (CON panel), we could not detect
P incorporation in the catalytic subunit of PKA,
indicating that autophosphorylation did not occur. It has been reported
that autophosphorylation of the catalytic subunit of PKA is inhibited
under these conditions (Geahlen et al., 1986). Nevertheless,
catalytic subunit autophosphorylation was observed in some experiments
(data not shown). In contrast, we consistently detected
autophosphorylation centered around 50 kDa in plasma membrane sample
(lane 2 of the CON panel). This phosphorylation spot
could be resolved in some studies as two autoradiographic bands that we
name together as 50-kDa protein kinase. When casein, a known substrate
for PKA (Geahlen et al., 1986), was added to the gel mixture
(upper CAS panel), two major in situ phosphorylations
spots were observed corresponding to the catalytic subunit of PKA
(approximately 41 kDa) and to the 50-kDa plasma membrane protein
kinase. The extent of
P incorporation was much higher than
in control gels and, therefore, much shorter exposures and less
sensitive films were needed. The autoradiographies shown corresponded
to 16-h exposure and high sensitivity film (CON panel) or 6 h
and low sensitivity film (upper CAS panel). This indicates
that casein is a substrate for both protein kinases. In some
experiments plasma membrane preparations also displayed an additional
protein kinase activity with the same apparent mobility as the
catalytic subunit of PKA that we named 41-kDa protein kinase and likely
is the catalytic subunit of PKA present in plasma membrane samples (see
lane 4 of the lower CAS panel). The detection with
casein of the 41-kDa protein kinase of plasma membrane depended, by and
large, on the sample batch and storage conditions. Shorter storage and
freshly assayed samples were usually needed to visualize the 41-kDa
membrane species. In contrast, when using rGP as a substrate, the
P incorporation in the position of the catalytic subunit
of PKA was surprisingly high in both samples and, therefore, very short
exposures of the gel (10 min) displayed patterns as the one shown in
rGP. This indicates that rGP is also a substrate for PKA and for the
41-kDa protein kinase of plasma membrane. Longer exposures to detect
the 50-kDa protein kinase activity were not reliable due to the masking
effect of the 41-kDa protein kinase activity. Gels containing less rGP
and the use of longer term stored plasma membrane samples, displaying
less 41-kDa protein kinase activity, were needed to visualize the
50-kDa protein kinase in the plasma membrane (upper rGP-0.1
panel). Elution and analysis of the phosphorylated material from
the substrate containing gels revealed that both protein kinases and
substrates are phosphorylated (data not shown). These data demonstrate
the existence of at least two protein kinases in human kidney plasma
membrane samples, one likely to be the catalytic subunit of PKA,
whereas the other shows different apparent molecular weight and
substrate preferences, indicating that it is indeed a distinct protein
kinase.
Figure 6:
Renaturation assays of protein kinases
from plasma membrane and from other sources after SDS-PAGE. In the
upper panels, 30 units (panels CON, CAS, rGP) or 10
units (panel rGP-0.1) of bovine PKA catalytic subunit from
Promega (lanes 1) or 40 µg of purified plasma membrane
from human cortex kidney (lanes 2) were analyzed by SDS-PAGE
in 10% gels polymerized in absence (CON panel) or presence of
1 mg/ml casein (CAS panel), 1 mg/ml rGP (rGP panel),
or 0.1 mg/ml rGP (rGP-0.1 panel). In lower panels (CAS and rGP-0.1), similar studies were carried
out with 15 µg of different samples representing the different
purification steps of plasma membrane from human cortex kidney: crude
homogenate (lanes 1), homogenate soluble material (lanes
2), homogenate membrane fraction (lanes 3), or saccharose
gradient-purified plasma membrane (lanes 4). After
renaturation and phosphorylation steps, the gels were fixed and free
[-
P]ATP rinsed out. Gels were then dried
and exposed at -70 °C for different time periods. From
left to right and from top to
bottom, the individual exposure times were: 16, 6, 0.16, 4, 3
and 0.5 h, respectively. The plasma membrane samples used in upper
panels were from -20 °C storage, whereas samples used in
lower panels were freshly prepared. In the later cases, the
samples from each purification step were stored at +4 °C until
use. In the anti-PKA panel, 25 µg of plasma membrane were
analyzed in a 8-18% gradient gel, transferred to Immobilon P
(Millipore) and blotted without (1) or with affinity-purified
antibodies against the catalytic subunit of PKA (2). Arrowheads indicate the positions, from top to bottom, of
the 50-kDa plasma membrane protein kinase and the PKA catalytic
subunit.
To further investigate the structural relation between these
two protein kinase activities and the plasma membrane, we carried out
similar renaturation studies with different human kidney cortex
fractions (lower panels of Fig. 6): crude homogenate
(lanes 1), homogenate soluble material (lanes 2),
homogenate membrane fraction (lanes 3), or purified plasma
membranes (lanes 4) using casein (lower CAS panel) or
recombinant antigen (lower rGP-0.1 panel) as substrates. The
50-kDa protein kinase associated to membrane fractions, and its
specific activity increased with plasma membrane purification,
indicating its plasma membrane origin (compare lane 1 with
lanes 3 and 4 of the lower CAS panel). These
results were consistent with Western blot analysis of similar samples,
using two different antibodies against two distinct plasma membrane
markers, low density lipoprotein receptor and
Na/K
-ATPase (data not shown). In
contrast, the 41-kDa protein kinase was detected in soluble as well as
in membrane fractions of homogenates (lanes 2 and 3 of the lower CAS and rGP-0.1 panels), and plasma
membrane purification did not increase its specific activity to the
same extent as for the 50-kDa protein kinase (compare lane 1 with lanes 3 and 4 in the lower CAS and
rGP-0.1 panels). This could reflect cytosolic PKA trapped in
the membrane fractions. However, the content of
glyceraldehyde-3-phosphate dehydrogenase, a cytosolic marker used in
parallel Western blot studies, was found to dramatically decrease upon
increased purification of the plasma membranes (data not shown). This
could indicate that in addition to the cytosolic form of PKA, there
exists a membrane-associated PKA that is mixed with the cytosolic form
in early purification steps. The presence of PKA in our plasma membrane
samples was further confirmed by Western blot analysis using
affinity-purified antibodies against the catalytic subunit of bovine
PKA (anti-PKA panel).
P
incorporation by each of the three protein kinases as observed after
removing the 26 amino-terminal residues strongly suggest that both
protein kinases found in human kidney plasma membrane samples are
catalytically of the PKA type and that the 41-kDa kinase likely is the
catalytic subunit of PKA. To further assess this, rGP and rGP-Ala9,26
were similarly assayed and the phosphorylated materials specifically
digested and immunoprecipitated (lanes 1 and 2 of
Fig. 7B, respectively). The lack of phosphorylation of
the amino-terminal region upon substitution of the two serines
conforming to PKA consensus sites supports the idea that 50-kDa protein
kinase is catalytically a type A-like protein kinase and that the
41-kDa kinase is indeed the catalytic subunit of PKA.
Figure 7:
Renaturation assays of protein kinases
from plasma membrane on Western blot membranes. In A, rGP
(lane 1), -26rGP (lane 2), and GPpep1 (lane
3) phosphorylated by Western blot-renatured 50-kDa plasma membrane
protein kinase were analyzed by reducing SDS-PAGE and autoradiography.
In B, similarly phosphorylated rGP (lane 1) and
rGP-Ala9,26 (lane 2) were reduced, digested, alkylated,
specifically immunoprecipitated with anti-GPpep1 polyclonal antibodies,
and analyzed as in A. Similar results were obtained when the
phosphorylating enzyme was Western blot-renatured PKA catalytic subunit
(Promega) or human kidney plasma membrane 41-kDa protein kinase.
Arrowheads indicate the positions, from top to
bottom, of rGP, -26rGP, and GPpep1 (A) and the
V8 digestion product of rGP and rGP-Ala9,26 specifically
immunoprecipitated (B).
3-containing collagen IV and that alternative
splicing may regulate this process through phosphorylation
independently of collagen IV synthesis and secretion (Quinones et
al., 1992; Bernal et al., 1993). Consistently,
recombinant proteins representing the antigen and its alternative forms
incorporate
P at very different rates in the presence of
PKA catalytic subunit indicating that alternative splicing serves, at
least in part, to regulate the amino-terminal
phosphorylation.
(
)
It has been shown that
phosphorylation of some extracellular proteins promotes RGD-mediated
cell attachment while dephosphorylation inhibits it (Ek-Rylander et
al., 1994 and references therein). In the case of the Goodpasture
antigen, if the RGD motif within the phosphorylatable region is
functional, phosphorylation of the adjacent Ser could also serve as a
mechanism to regulate cell adhesion to basement membrane. Native
collagenous domains bind integrins through a distinct motifs and
RGD-dependent cell binding sites are generated in these domains only
after denaturation (Wayner and Carter, 1987; Aumailley et al.,
1989; Vandenberg et al., 1991; Gullberg, et al.,
1992; Eble et al., 1993; Pfaff et al., 1993). This
strongly suggests that geometrical restrictions imposed by triple helix
secondary structure hinder favorable RGD configuration on the surface
of the protein preventing effective integrin binding. Therefore, an RGD
in a purely triple-helical environment would likely be a bad candidate
for cell binding through integrins. However, due to the positioning of
the RGD-containing region studied here at the junction between triple
helix and NC1 domains and to the highly hydrophilic nature of this
region, its secondary structure could not be a perfect triple helix but
rather a transition structure where the presence of a phosphate group
might fully expose the RGD, making it accessible to cell receptors.
. One of them appears to be the catalytic subunit of
PKA. The other, although phosphorylating the recombinant antigen at the
same sites as the catalytic subunit of PKA, displays different apparent
molecular weight, is only found in membrane fractions and shows
different substrate affinities. Consistently, GPpep1 is phosphorylated
in a cAMP-dependent fashion by plasma membrane fractions obtained by
affinity chromatography using Sepharose 4B-GPpep1.
(
)
In some renaturation studies, we found an additional
phosphorylating activity associated with the plasma membrane migrating
in the high molecular weight range. This activity was not always
present and its
P incorporation did not increase with the
presence of any of the above substrates. The existence of additional
protein kinases, which do not renature after SDS-PAGE but can
specifically phosphorylate the Goodpasture antigen, cannot be ruled
out.
Table:
Phosphorylation of Goodpasture peptides by
several protein kinases
P incorporation. +Ca
and +PL
denote the presence in the assay mixture of 100 µM CaCl
or 5 µg/ml phosphatidylserine and 0.4
µg/ml diacylglycerol, respectively. Each assay was performed in
duplicate, and each value represents the average of at least two
experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.