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Department of Medicine, Division of Nephrology and Kidney Disease Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
1 To whom requests for reprints should be addressed at Medical College of Wisconsin, Department of Medicine/Kidney Disease Center, 8701 Watertown Plank Rd., Milwaukee, WI 53226. E-mail: sorokin{at}mcw.edu
Abstract
Endothelin-1 (ET-1) effects in human glomerular mesangial cells (GMC) include proliferation, contraction, and extracellular matrix synthesis. Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human glomerulae. Working in concert with Pyk2, adaptor protein CrkII and a recently discovered guanidine exchange factor for certain small GTPases BCAR3 can be involved in ET-1 signaling in the kidney. Signaling through CrkII and BCAR3 might be critical in some proliferative kidney pathologies. The current study was designed to determine the possibility of CrkII and BCAR3 interaction in response to ET-1 in human GMC and the role of Pyk2 in the association of these proteins. Using adenovirus-mediated transfer of genes encoding either green fluorescent protein (control) or dominant interfering Pyk2 construct, we demonstrated that CrkII and BCAR3 can be coprecipitated from unstimulated and ET-1 stimulated GMC; ET-1 treatment time-dependently increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to a significant decrease in CrkII/BCAR3 association both basal and stimulated.
Key Words: BCAR3 • CrkII • endothelin • human glomerular mesangial cells • Pyk2
Introduction
Endothelin-1 (ET-1) is a strong vasoconstrictor peptide participating in the development of different types of kidney pathology (1, 2). ET-1 stimulation leads to contraction, proliferation, and induction of profibrotic extracellular matrix synthesis in glomerular mesangial cells (GMC) (3–8), directly decreasing glomerular filtration rate and contributing to sclerotic lesion formation (9). There are two types of G-protein–coupled ET-1 receptors, ETA and ETB, enabling the peptide effects in GMC. ET-1 receptor activation evokes a wide variety of intracellular signaling events.
Calcium-regulated nonreceptor, proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of ET-1 signaling in human GMC. Pyk2 activation provides a link between G-protein–coupled receptors and the induction of tyrosine phosphorylation via mobilization of intracellular calcium (10–14). We and others demonstrated that in different cell types Pyk2 can play a crucial role in ET-1–mediated cytoskeleton transformation, including migration, contraction, and adhesion (10, 15–19). The focal adhesion-dependent p130Cas/Crk/Pyk2/c-Src–mediated pathway is selectively involved in ET-induced JNK MAP kinase activation in cardiomyocytes (20).
CrkII is a member of the family of adaptor-type signaling molecules that consist mostly of SH2 and SH3 domains. Other members of CrkII family of adaptor proteins are v-Crk, CrkI, CrkIII, and CrkL (21, 22). Crk-family proteins are involved in a variety of signaling cascades, such as those induced by growth factors receptors, integrin molecules, T-cell receptors, B-cell antigen receptors, and cytokines. However, the role of CrkII in ET-1 signaling still remains uncertain. It has been postulated that the primary function of Crk is to recruit cytoplasmic proteins to the vicinity of tyrosine kinases through SH2-phosphotyrosine interaction. Thus the output from Crk depends on the SH3-binding proteins, which include the tyrosine kinase Abl, the C3G guanine nucleotide exchange factor for Rap1, DOCK180 for Rac, and Sos for Ras. In addition, another guanidine exchange factor for certain G-proteins, BCAR3 (its murine homolog is known as AND-34), can be indirectly engaged in CrkII-dependent transduction via p130Cas scaffolding protein, which has been shown to interact with BCAR3 (23, 24). Hypothetically, the BCAR3 proline-rich site and phosphotyrosines can directly associate with CrkII SH3 and SH2 domains, respectively. Despite the potential importance of BCAR3 in proliferation cascades, the role of BCAR3 in ET-1–induced signaling remains obscure.
The current study was designed to examine the possibility that CrkII and BCAR3 interact in response to ET-1 in human GMC and to investigate the role of Pyk2 in this association. We demonstrated that CrkII and BCAR3 can be coprecipitated from GMC both before and after stimulation with ET-1; ET-1 treatment increased CrkII/BCAR3 complex formation; and inhibition of endogenous Pyk2 autophosphorylation led to significant decrease in CrkII/BCAR3 association.
Materials and Methods
Materials.
Purified human ET-1 was from Calbiochem (San Diego, CA). The enhanced chemiluminescence detection system was supplied by Amersham Pharmacia Biotech (Baie d’Urfé, Quebec, Canada). The BCA protein assay kit was from Pierce (Rockford, IL). All other reagents, unless otherwise indicated, were from Sigma (St. Louis, MO).
Antibodies.
To perform immunoprecipitation we used mouse monoclonal anti-human Crk from BD Transduction Laboratories (Lexington, KY), and control mouse IgG antibodies from BD Biosciences Pharmigen. For Western blotting, rabbit polyclonal anti-Pyk2 and anti-Crk and mouse monoclonal anti-FLAG antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-actin antibody was from ICN Biochemicals (Costa Mesa, CA). Polyclonal phosphorylation state-specific anti-Pyk2 Tyr-402 antibody was from Biosource International (Camarillo, CA). Previously described anti-human BCAR3 rabbit serum (25) was a gift from Dr. Adam Lerner (Boston University School of Medicine, Boston, MA). The horseradish peroxidase–E conjugated goat anti-mouse and goat anti-rabbit immunoglobulins were from Bio-Rad (Hercules, CA).
Cell Culture.
All materials for cell culturing were purchased from Invitrogen (Carlsbad, CA). Previously characterized SV40-transformed human GMC (26) was cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml) in a 37°C humidified incubator with 5% CO2.
Adenovirus-mediated Gene Transfer.
The adenoviral vector (Ad), encoding the carboxyl terminus of Pyk2, termed CRNK for calcium-dependent tyrosine kinase-related non-kinase (AdCRNK), was constructed from the replication-deficient adenovirus type 5 with deletions in the E1 and E3 genes as previously described (16). GMC, starved for 24–48 hrs, were incubated either with adenovirus encoding Green Fluorescent protein (AdGFP) or AdCRNK at a multiplicity of infection of 40 plaque-forming units/cell for 1 hrs at 37°C with periodic shaking, followed by the addition of the medium supplemented with 0.5% serum. Forty-eight hours later, cells were prestarved in serum-free media for 3 hrs, stimulated with ET-1, and lysed for further analysis. Efficiency of gene transfer in GMC was 80%–100%, as determined by fluorescence microscopy visualization of AdGFP control.
Cell Lysis, Immunoprecipitation, and Immunoblot Analysis.
After stimulation with ET-1 (100 nM) for a given period, the cells were lysed as described previously (27). Briefly, cells were washed twice with ice-cold tris-buffered saline (TBS) after the treatment. They were then lysed for 20 mins at 4°C in 800 µl of lysis buffer containing the following: 10 mM Tris HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 30 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 10 mM sodium fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Cell lysates were collected in microcentrifuge tubes and cleared by centrifugation at 14,000 g for 8 mins at 4°C. Equalized for protein content, the supernatants were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for Western blot analysis (20–30 µg) or subjected to immunoprecipitation (700–800 µg).
Cells lysates were immunoprecipitated with either 2 µg antibodies against CrkII, or control mouse IgG for 1.5 hr at 4°C with constant rotation. Protein A Sepharose (40 µl; Roche, Basel, Switzerland) was added, and the lysates were incubated for an additional 1 hr. After washing the immunoprecipitates three times in the lysis buffer, 3x Laemmli sample buffer was added, and the samples were boiled for 5 mins, subjected to SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After transfer of proteins, the polyvinylidene difluoride membrane was blocked with TBS containing 0.1% Tween 20 (TBS-T) and 3% bovine serum albumin (United States Biochemical, Cleveland, OH) for phosphorylated tyrosine-specific primary antibody, or TBS-T and 5% nonfat dry milk for all others for 2 hrs at room temperature. Primary antibodies were added, and the membranes were incubated overnight at 4°C with constant rotation. The membranes were then washed three times with TBS-T before the addition of secondary antibodies. Corresponding goat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase conjugates antibodies were diluted 1:3000 in TBS-T containing 5% nonfat dry milk, and incubated with the membrane for 1 hr at room temperature. The membranes were then washed three times with TBS-T before development of immunoreactive signal by enhanced chemiluminescence reagent.
Results
Cell lysates from AdGFP and AdCRNK transfected cells were stimulated for 1, 2, and 6 mins with 100 nM ET-1 and subjected to either direct SDS-PAGE followed by immunoblot (IB) analysis or immunoprecipitation (IP) with anti-Crk antibody and then IB. We observed coprecipitation CrkII with BCAR3 in GMC (Fig. 1
, first panel, top). Interaction between these two proteins was significantly increased at 2 and 6 mins after ET-1 stimulation compared with the basal level.
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(fourth and fifth panels, top), adenovirus-mediated CRNK expression caused significant reduction in Pyk2 autophosphorylation in response to ET-1 application without significant effect on total Pyk2 protein amount. In parallel, we observed noticeable diminishing of CrkII/BCAR3 complex formation in CRNK-expressing GMC with or without ET-1 treatment compared to GFP-expressing controls (Fig. 1, first panel). Equal amounts of BCAR3 were expressed in all experimental settings as detected by IB with anti-BCAR3 antibody (Fig. 1, third panel). It has previously been shown that Pyk2 does not directly interact with CrkII adaptor protein (28), and we did not observe any coprecipitation of Pyk2 with CrkII in our experiments (data not shown). However, we cannot exclude the possibility that CRNK, via its proline rich sequences, can interfere with CrkII/BCAR3 assembly by directly binding to the SH3 domain of CrkII. To check this hypothesis, we conducted the next experiment.
Cell lysates from AdGFP- and AdCRNK-infected cells were stimulated for 2 and 6 mins with ET-1 and subjected to SDS-PAGE followed by IB or IP with anti-Crk antibody followed by SDS-PAGE and IB analysis. On ET-1 stimulation, CrkII was phosphorylated, as shown by the upward shift of CrkII band in total cell lysates IB (Fig. 2, second panel, top; 2 and 6 mins comparison to prestimulation level). Expression of CRNK did not affect CrkII activation in response to ET-1. Immunoprecipitation experiments resulted in the equal amount of CrkII protein collected from each time point (Fig. 2, second panel, top). No significant CRNK and CrkII association had been detected in IP samples, despite strong CRNK expression in GMC as revealed by IB with anti-FLAG antibody (Fig. 2, first panel).
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The glomerular mesangial cell is the target of both immune and nonimmune forms of injury in many diverse glomerular diseases. In response to injury, mesangial cells may undergo several scenarios, including contraction, proliferation, increased extracellular matrix synthesis, and apoptosis. These cellular responses may lead to progressive decrease in glomerular filtration rate and to glomerular scleralization. ET-1 is one of the significant para- and autocrine factors that regulate GMC homeostasis during development of kidney function abnormalities (1, 2). However, details of ET-1–induced cellular responses in glomerulae remain obscure. Pyk2 is an important non-receptor tyrosin kinase that mediates ET-1 signaling. Pyk2-mediated activation of intracellular cascades contributes to pathologic intracellular events in the kidney during hypertension and inflammatory disease, which are often accompanied by augmented GMC proliferation. Regulated by adhesion, inflammatory cytokines and ET-1, a BCAR3-involving complex, might also be important during mesangial proliferative kidney disease such as diabetic glomerulosclerosis or proliferative glomerulonephritis.
The present work is the first demonstration that CrkII interacts with BCAR3 and that ET-1 triggers the increase in CrkII/BCAR3 complex formation. We have also observed that activation of Pyk2 influenced CrkII/BCAR3 association without direct CRNK interaction with CrkII. Phosphorylation of Pyk2 and CrkII occurred independently of each other, because CRNK expression did not inhibit CrkII phosphorylation. Most likely, Pyk2 phosphorylation may play a role in ET-1–induced modifications of BCAR3 or CrkII translocation. Theoretically, SH2 and proline-rich domains of BCAR3 can participate in the protein-protein contact with phosphorylated tyrosines and SH3 domain of CrkII, respectively. In agreement with our speculation, the SH2 domain of BCAR3 was demonstrated to be important for activation of phosphatidylinositol 3-kinase (29). Also, our data suggest that an additional protein, presumably phosphorylated by Pyk2, might be required for the complex assembly. One of the possible candidates to link Pyk2 activation with BCAR3 and CrkII interaction might be the scaffolding protein p130Cas. In particular, p130Cas can be a substrate for Pyk2 and can form stable complex with CrkII (30). Furthermore, BCAR3 interacts via its C-terminal GEF domain and additional 70 amino acids with some unique sequence in p130Cas (23, 24). However, the exact mechanism of CrkII/BCAR3 complex assembling requires further experimental elucidation.
The hypothetical physiologic role of Pyk2-dependent CrkII/BCAR3 association may be the regulation of BCAR3 GEF activity for small GTPases, including Cdc42, Rac1, Ral, Rap1, and R-Ras (24, 31). In transmitting signals downstream to the cytoskeleton-dependent adhesion, contraction, proliferation, and migration, small G-proteins rotate between GDP- and GTP-bound forms. Activation of a particular small G-protein by BCAR3 may be a cell-specific event. Which particular GTP/GDP-binding molecule is employed, as a result of Pyk2-dependent CrkII/BCAR3 assemblance, needs to be determined. Alternatively, ET-1–induced Pyk2-dependent CrkII/BCAR3 cascade can be linked to the proliferation pathway involving activation of nuclear factor-
B (NFB) (32). The NFB family comprises ubiquitously expressed transcription factors that are known to be critical regulators of mammalian immune and inflammatory responses, proliferation, and more recently have been shown to associate with chemotherapy resistance in parallel with BCAR3. NfB activation can provide a basis for the long-term ET-1 effects in kidney. However, the experimental examination of CrkII/BCAR3 complex physiologic importance is beyond the scope of our current study.
In summary, we have demonstrated that in human GMC ET-1, through Pyk2 kinase, induces association of adaptor protein CrkII with guanine nucleotide exchange factor BCAR3. The signaling complex described here may have short- and long-term pathophysiologic significance during development of proliferative renal diseases. Better understanding of postreceptor signaling through protein-protein interaction and tyrosine phosphorylation triggered by ET-1 can open beneficial approaches to intervention in the activation of selective pathways mediating mesangial cell contraction, hypertrophy, and proliferation.
Acknowledgments
We are grateful to Dr. Adam Lerner (Boston University School of Medicine, Boston, MA) for the generous gift of anti-BCAR3 rabbit serum.
Footnotes
This study was supported by the National Institutes of Health research grants DK 41684 and HL 22563 to Dr. A. Sorokin and by Postdoctoral Fellowship from AHA 0525801Z to Dr. V. (Petrukhina) Rufanova.
Received for publication September 29, 2005. Accepted for publication November 23, 2005.
References
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-actin antibodies (lower panel).