©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of the Goodpasture Antigen by Type A Protein Kinases (*)

Fernando Revert (1)(§), José R. Penadés (1)(¶), Mara Plana (2), Dolores Bernal (1) (3)(**), Charlott Johansson (4), Emilio Itarte (2), Javier Cervera (1), Jorgen Wieslander (4), Susan Quinones (5), Juan Saus (1) (3)(§§)

From the (1) Fundación Valenciana de Investigaciones Biomédicas, Instituto de Investigaciones Citológicas, 46010 València, Spain, the (2) Departament de Bioqumica i de Biologa Molecular, Facultat de Ciències, Universitat Autnoma de Barcelona, 08193 Bellaterra, Barcelona, Spain, the (3) Departament de Bioqumica i Biologa Molecular, Facultat de Farmacia, Universitat de València, 46100 Burjassot, València, Spain, the (4) Department of Nephrology, University Hospital, S-221 85 Lund, Sweden, the (5) Department of Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Collagen IV is the major component of basement membranes. The human 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.


INTRODUCTION

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 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.

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 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.


EXPERIMENTAL PROCEDURES

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 (3(IV) NC1 Monomers) from Human and Bovine Kidneys

The 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 HPO and assay of the radioactivity. K values were estimated using the Enzfitter adjusting program (Leatherbarrow, 1987).

Recombinant antigens (22 µg/ml) were similarly phosphorylated with 9 units of PKA catalytic subunit (Promega) and 10 µCi of [-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.

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 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% HO 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.


RESULTS

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 ().

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 (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).

To further characterize the phosphorylation of GPpep1 by PKA, the reaction mixture was applied to an HPLC µBondapack 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, KGKRGDSGSPATWTTRGFVFT, 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 (KGKRGDSG) 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).

These data strongly suggest that the Goodpasture antigen is phosphorylatable at Ser by PKA in the region where maximum differences exist between the different NC1 domains of collagen IV chains.

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 (GLKGKRGDSGSPATWTTRGFVFTRHS) 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.

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 (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).

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 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).




DISCUSSION

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 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.

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. 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.

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.

  
Table: Phosphorylation of Goodpasture peptides by several protein kinases

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 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.



FOOTNOTES

*
This work was supported by Grant 89-0406 from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS, Spain) and Grants PB87/0951 and SAL91/0513 from the Comisión Interministerial de Ciencia y Tecnologa (CICYT, Spain) (to J. S.), CICYT Grant PB89/0330 (to E. I.), National Institutes of Health Grant DK-42514 (to S. Q.), and Grant 16X-09487 from the Swedish Medical Research Council and the Medical Faculty, University of Lund, Sweden (to J. W.). Additional support came from National Institute of Environmental Health Science Center of Excellence Grant ESO 5022. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Consellera de Educació i Ciencia of Comunidad Valenciana.

Recipient of a fellowship from the Formación de Personal Investigador del Ministerio de Educación y Ciencia from Spain.

**
Recipient of a fellowship from the Fondo de Investigaciones Sanitarias de la Seguridad Social and currently is Profesor Ayudant del Departament de Bioqumica i Biologa Molecular of Universitat de Valéncia.

§§
To whom correspondence should be addressed: Instituto de Investigaciones Citológicas, C/Amadeo de Saboya 4, 46010 Valencia, Spain. Tel.: 34-6-3698500; Fax: 34-6-3601453.

The abbreviations used are: NC1, non-collagenous domain; HPLC, high performance liquid chromatography; rGP, recombinant Goodpasture antigen; PCR, polymerase chain reaction; PKA, cAMP-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis.

The position of amino acids in the Goodpasture antigen are numbered from the putative collagenase cleavage site (Quinones et al., 1992).

J. R. Penadés, F. Revert, and J. Saus, manuscript in preparation.

M. Plana, F. Revert, E. Itarte, and J. Saus, unpublished results.


ACKNOWLEDGEMENTS

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.


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