Epidermal Growth Factor Activates Na+/H+ Exchanger in Podocytes Through a Mechanism That Involves Janus Kinase and Calmodulin

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Biochimica et Biophysica Acta 1793 (2009) 1174–1181

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Epidermal growth factor activates Na+/H+ exchanger in podocytes through a mechanism that involves Janus kinase and calmodulin

Sonya D. Coaxum, Maria N. Garnovskaya, Monika Gooz, Aleksander Baldys, John R. Raymond ⁎

Medical and Research Services of the Ralph H. Johnson VA Medical Center, USA Division of Nephrology, Department of Medicine, of the Medical University of South Carolina, Charleston, South Carolina 29425, USA

article info abstract

Article history: Sodium-proton exchanger type 1 (NHE-1) is ubiquitously expressed, is activated by numerous growth Received 24 September 2008 factors, and plays signiﬁcant roles in regulating intracellular pH and cellular volume, proliferation and Received in revised form 28 February 2009 cytoskeleton. Despite its importance, little is known about its regulation in renal glomerular podocytes. In the Accepted 19 March 2009 current work, we studied the regulation of NHE-1 activity by the epidermal growth factor receptor (EGFR) in Available online 31 March 2009 cultured podocytes. RT-PCR demonstrated mRNAs for NHE-1 and NHE-2 in differentiated podocytes, as well as for EGFR subunits EGFR/ErbB1, Erb3, and ErbB4. EGF induced concentration-dependent increases in Keywords: ﬂ Cytosensor microphysiometer proton ef ux in renal podocytes as assessed using a Cytosensor microphysiometer, were diminished in the ECAR presence of 5-(N-methyl-N-isobutyl) amiloride or in a sodium-free solution. Furthermore, pharmacological Phosphorylation inhibitors of Janus kinase (Jak2) and calmodulin (CaM) attenuated EGF-induced NHE-1 activity. Co- AG1478 immunoprecipitation studies determined that EGF induced formation of complexes between Jak2 and CaM, as well as between CaM and NHE-1. In addition, EGF increased levels of tyrosine phosphorylation of Jak2 and CaM. The EGFR kinase inhibitor, AG1478, blocked activation of NHE-1, but did not block EGF-induced phosphorylation of Jak2 or CaM. These results suggest that EGF induces NHE-1 activity in podocytes through two pathways: (1) EGF→EGFR→Jak2 activation (independent of EGFR tyrosine kinase activity)→tyrosine phosphorylation of CaM→CaM binding to NHE-1→conformational change of NHE-1→activation of NHE-1; and (2) EGF→ EGFR→ EGFR kinase activation→ association of CaM with NHE-1 (independent of Jak2)→conformational change of NHE-1→activation of NHE-1. © 2009 Elsevier B.V. All rights reserved.

1. Introduction investigation because they have been implicated as both targets and instigators of renal injury in various progressive renal diseases Epidermal growth factor (EGF) plays a number of key roles in the [18,20–23]. Therefore, it would be desirable to elucidate signaling kidney, contributing to cellular proliferative and survival pathways, pathways in podocytes, which may be important in delineating the renal metabolism [1], regenerative hyperplasia [2], tubulointerstitial mechanisms through which podocytes contribute to the progression injury [3], tubulogenesis [4], transport [5], renal cyst formation [6] and of glomerular injury. renal development [7]. EGF also has been implicated in the genesis Because of the emerging role of the sodium-proton exchanger and development of polycystic kidney disease [8,9]. Despite the type 1 (NHE-1), also known as product of SLC9A1, solute carrier importance of EGF in many renal functions, particularly in renal family 9A, type 1 in the regulation of the cytoskeleton [24], apoptosis tubules and mesangial cells, little is known about its effects in and cellular proliferation [25], cell cycle control [26], and develop- glomerular podocytes. Podocytes are critical for the maintenance of ment and maintenance of the transformed cellular phenotype [27], normal glomerular structure [10,11], and aberrant podocyte function we thought that it might be of interest to develop a better has been implicated in the pathogenesis of chronic renal diseases understanding of its regulation in podocytes. Previous studies have [12–17]. These highly specialized cells are characterized by the demonstrated that EGF stimulates NHE-1 in non-renal cells [28–30], formation of foot processes that are interconnected by the slit but the signaling pathways involved in the regulation have not been diaphragm, which is a critical component of the glomerular ﬁltration fully elucidated. Moreover, currently there are no data on the barrier [18,19]. Podocytes are emerging as the focus of intense regulation of NHE-1 in podocytes. In that regard, recent studies from our laboratory have shown that NHE-1 can be activated by G protein-coupled receptors or hypertonic medium, through Janus ⁎ Corresponding author. Medical University of South Carolina, Colcock Hall, Ofﬁce of kinase 2 (Jak2)-dependent phosphorylation of CaM, and subsequent the Provost, 179 Ashley Avenue, Charleston, SC 29425, USA. Tel.: +1 843 792 3031; fax: – +1 843 792 5110. interaction between CaM and NHE-1 [31 33]. Therefore, we wanted E-mail address: [email protected] (J.R. Raymond). to determine whether EGF is important for regulating NHE-1 activity

0167-4889/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2009.03.006 S.D. Coaxum et al. / Biochimica et Biophysica Acta 1793 (2009) 1174–1181 1175 in podocytes, and to identify key components of the signal transcripts were identified using SuperArray's Multigen-12 RT-PCR transduction pathway linking EGF and NHE-1. In the current study, profiling kit. To analyze NHE-1 message an already published primer we demonstrate that EGF stimulates NHE-1 activity in podocytes. In pair was used: 5′-TCTGCCGTCTCAACTGTCTCTA-3′ sense, 5′- addition, we have shown that Jak2 and CaM play critical roles in the CCCTTCAACTCCTCATTCACCA-3′ antisense [37], which generated a activation of NHE-1 by EGF in podocytes. 422 base pair (bp) product. For analyzing other NHE messages, new PCR primer pairs were designed: NHE-2 (5′-ACACACAACATCCGGGT- 2. Materials and methods CATT-3′ sense, 5′-CGCTTGTGTCTGCCGTCA-3′ antisense) resulted in a 982 bp product; NHE-3 (5′-CGCCTGGAGTCCTTTAAGTC-3′ sense, 5′- 2.1. Cell culture GGAGAACACGGGATTATCAAT-3′ antisense) generated a 294 bp product, and NHE-4 (5′-GAGGACATAGAAGCGGTGGAC-3′ sense, 5′- The podocyte cell line was kindly provided by Dr. Peter Mundel of AGGAGAAAGCCGCTTGATTC-3′ antisense) resulted in a 993 bp PCR Mt. Sinai School of Medicine. Podocytes were cultured as previously product. Target specificities of the PCR primers were confirmed by described [34]. Undifferentiated podocytes were maintained in RPMI- sequencing the PCR products using the MUSC sequencing core facility. 1640 medium containing 10 U/ml of mouse recombinant γ- interferon, 10% FBS, 100 U/ml of penicillin and 100 μg/ml of 2.5. Immunoprecipitation streptomycin at 33 °C in 95% air and 5% CO2. To induce differentiation, podocytes were maintained in the same medium as undifferentiated For immunoprecipitation of Jak2 and NHE-1, quiescent differ- podocytes without γ-interferon at 37 °C in 95% air and 5% CO2 for entiated podocytes grown on 100 mm collagen-coated tissue culture 14 days. All experiments were conducted using differentiated dishes were pretreated with 50 μM of AG490 or 20 μM of AG1478 for podocytes, unless stated otherwise. 30 min prior to treatment with 10 ng/ml of EGF or vehicle for 5 min, and then lysed in 1 ml/dish of RIPA buffer (50 mM Tris–HCl, pH 7.4, 2.2. Immunofluorescence microscopy 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA) supplemented with protease inhibitors (1 mM AEBSF, 0.8 μM Immunolabeling was performed as previously described [34]. Cells aprotinin, 50 μM bestatin, 15 μM E-64, 20 μM leupeptin, 10 μM were seeded in 35-mm collagen-coated glass-bottom culture dishes pepstatin A, 1 mM sodium orthovanadate, 1 mM sodium fluoride and (MatTek Corporation) and fixed with 2% paraformaldehyde, 4% 1 mM PMSF). Equal amounts of proteins (1.5 mg) were precleared by sucrose in phosphate-buffered saline (PBS) for 10 min at room incubation with protein A/G sepharose beads for 30 min at 4 °C. temperature. Subsequently, cells were permeabilized with 0.3% Triton After a brief centrifugation, the supernatants were removed and X-100 (Sigma) in PBS for 5 min, following which nonspecific binding incubated with either agarose conjugated anti-JAK2 antibody sites were blocked with 2% fetal calf serum, 2% BSA and 0.2% gelatin in (Upstate) or anti-NHE-1 antibody (Chemicon) overnight at 4 °C. PBS for 1 h. Incubations with the appropriate dilutions of primary and Immunoprecipitates were captured with 50 μl of protein A/G beads secondary antibodies (as directed by the manufacturer) were at 4 °C for 1 h. Then, the samples were centrifuged and washed performed in blocking solution. The primary and secondary antibodies used were: anti-WT1 (1:50) (Santa Cruz Biotechnology); anti- synaptopodin (Research Diagnostics, Inc.) and Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes). Confocal microscopy was performed using a Zeiss LSM 510 META laser-scanning microscope (Carl Zeiss, Inc.).

2.3. Microphysiometry

NHE-1 activity studies were conducted on a Cytosensor microphysiometer as previously described for other cell types [32,35,36]. Cells were plated on transwell membranes (3 μm pore size, 12 mm size) at a density of 300,000 cells per insert, and serum starved overnight on the day prior to experimentation. On the day of the experiment, the cells were washed with serum-free, bicarbonate-free F-12 medium, prior to being placed into microphysiometer chambers. The chambers were perfused at 37 °C with serum-free media or balanced salt solutions. After establishment of a stable baseline for at least five cycles, cells were exposed to the drugs for 4 cycles (360 s). Podocytes had low basal proton efflux levels (−10 to −40 μV/s), which roughly corresponds to millipH units/minute according to the Nernst equation). The extracellular acidification rate (ECAR, rate of accumulation of protons during a stop-flow period) was measured at peak stimulation after initiation of drug treatment, as is standard for microphysiometry studies. This typically occurred after two or three cycles (180 or 270 s) of exposure to EGF. Rate data were expressed as percentages of baseline values.

2.4. RT-PCR and PCR

RNA was prepared from differentiated and undifferentiated Fig. 1. Immunofluorescence analysis of podocyte markers. Cells were fixed and then stained for synaptopodin or WT-1 to confirm differentiated from undifferentiated podocytes using Trizol reagent following the manufacturer's protocol podocytes. Undifferentiated podocytes did not stain for synaptopodin (A). In contrast, fi (Invitrogen). Five micrograms of total RNA were used for rst strand differentiated podocytes stained for synaptopodin (B). Undifferentiated and differ- cDNA synthesis (SuperScript III RT-PCR kit, Invitrogen). EGFR/ErbB entiated podocytes stained for WT-1 (C and D). 1176 S.D. Coaxum et al. / Biochimica et Biophysica Acta 1793 (2009) 1174–1181

3. Results

3.1. Immunohistochemical conﬁrmation of podocyte differentiation

Podocytes were stained for WT-1 and synaptopodin. Undiffer- entiated podocytes did not stain for synaptopodin (Fig. 1, Panel A); however, the cells did stain for WT-1 (Fig. 1, Panel C). Differentiated podocytes stained for synaptopodin (Fig. 1, Panel B) and WT-1 (Fig. 1, Panel D). The results of the staining conﬁrm that in our hands, the cultured podocytes showed hallmarks of differentiation.

3.2. EGFR mRNAs are expressed in podocytes

Epidermal growth factor (EGF) receptors constitute a family of four prototypical receptor tyrosine kinases (ErbB1-4). EGF receptor (EGFR) subunits dimerize upon ligand binding, resulting in the formation of activated receptors. We determined which EGFR subunit mRNAs were expressed in podocytes using RT-PCR. Undifferentiated podocytes expressed the mRNAs for EGFR/ErbB1, Neu/HER2, ErbB3, and ErbB4 (Fig. 2A). Differentiated podocytes expressed the mRNAs for EGFR/ErbB1, Erb3, and ErbB4. Neu/HER2 mRNA was detectable at very minute levels in differentiated podocytes (Fig. 2A).

3.3. EGF induces concentration-dependent increases in ECAR

Fig. 2. Presence of functional EGFR in podocytes. (A) RT-PCR analyses of EGFR/ErbB Having established that podocytes express EGFR mRNAs, we next receptor subunit mRNA expression. Expression of different EGFR subunits in determined whether the cells expressed functional EGFR. We undifferentiated and differentiated podocytes is shown: EGFR (183 bp), Neu/HER2 fi (130 bp), ErbB3 (153 bp) and ErbB4 (204 bp). (B) Microphysiometry was used to measured EGF-induced increases in extracellular acidi cation rates analyze the concentration-dependent effects of EGF on extracellular acidification using microphysiometry under stop-flow conditions. Fig. 2B shows rate (ECAR) in podocytes. Independent experiments were performed a minimum that EGF increased proton efflux in a concentration-dependent of three times in triplicate, and the data are presented as mean± SEM of peak values.

thrice with 1 ml of RIPA buffer, and the proteins were eluted from the beads using 2× Laemmli sample buffer. Samples subsequently were separated by SDS-PAGE and transferred to PVDF membrane. Blots were probed with anti-calmodulin antibody (Upstate), and, to ensure equal NHE-1 and Jak-2 precipitation from the samples, with NHE-1 monoclonal antibody (Chemicon) or Jak-2 antiserum (Upstate). For phosphotyrosine immunoprecipitation experiments, quiescent podocytes grown onto 100 mm collagen-coated tissue culture dishes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM) or vehicle for 30 min, then stimulated with 10 ng/ml EGF or vehicle for 5 min and lysed in 0.5 ml/100 mm dish of RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1%

Nonidet P-40, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfo- nyl fluoride, aprotinin, leupeptin, and pepstatin at 1 μg/ml each). Cell lysates were precleared by incubating with protein A-agarose bead slurry for 30 min at 4 °C. Precleared lysates (1 μg/μl total cell protein) were incubated with monoclonal anti-phosphotyrosine antibody conjugated to protein A-agarose (Cell Signaling) overnight at 4 °C. The agarose beads were collected by centrifugation, washed twice with RIPA buffer and once with PBS, resuspended in 2× Laemmli sample buffer, boiled for 5 min, and subjected to SDS- PAGE and subsequent immunoblot analyses with polyclonal anti- phosphotyrosine, anti-EGFR, anti-Jak2, or with monoclonal anti- Fig. 3. EGF stimulates NHE-1 activity in podocytes. (A) RT-PCR analysis of NHE CaM antibodies (Upstate). isoforms. Expression of different NHE isoform mRNAs in (a) whole mouse kidney lysates; (b) undifferentiated podocytes; and in (c) differentiated podocytes are 2.6. Statistical analysis shown: NHE-1 (422 bp), NHE-2 (982 bp), NHE-3 (294 bp) and NHE-4 (993) bp. (B) Microphysiometry was used to characterize EGF-induced increases in ECAR. Podocytes were stimulated with 10 ng/ml of EGF in the presence or absence of Data were analyzed by paired, two-tailed Student's t test and sodium-free medium or MIA (a NHE-1 inhibitor). Values depicted are mean±SEM of b analysis of variance using GraphPad Statistics Software. P values 0.05 peak values from 3 independent experiments performed in triplicate. ⁎⁎Pb0.01 vs were considered significant. drug alone samples. S.D. Coaxum et al. / Biochimica et Biophysica Acta 1793 (2009) 1174–1181 1177 manner, confirming the presence of functional EGFR in differentiated podocytes.

3.4. EGF activates Na+/H+ exchange in podocytes

We next sought to determine the nature of the proton efflux pathway activated by EGF. Because EGF has been shown to stimulate sodium-proton exchangers in fibroblasts, esophageal epithelia and chondrocytes [30,38–40], we studied the expression of mRNAs encoding plasma membrane localized sodium-proton exchangers NHE-1, NHE-2, NHE-3, and NHE-4. Fig. 3A shows that differentiated podocytes express mRNA for NHE-1 and NHE-2, with the levels of NHE-1 mRNA predominating. Undifferentiated podocytes express only the mRNA for NHE-1 (Fig. 3A). The mRNAs for NHE-3 and NHE-4 were not detected in undifferentiated or differentiated podocytes. Thus, it is possible that EGF-mediated proton efflux from differentiated podocytes involves NHE-1 or NHE-2. In order to test the involvement of sodium-proton exchangers in the stimulation of proton efflux by EGF, we isotonically substituted tetramethylammonium (TMA) for sodium in the extracellular perfu- sate, thereby removing the extracellular substrate for sodium-proton exchangers. Fig. 3B shows that EGF stimulated proton efflux in a medium containing sodium, and that this effect was nearly abolished

Fig. 5. EGF induces formation of signaling complexes between Jak2 and CaM, and NHE-1 and CaM in podocytes. Co-immunoprecipitation experiments were performed as described under Materials and methods. (A) Podocytes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM), or vehicle for 30 min, and then stimulated with 10 ng/ml of EGF for 5 min prior to being immunoprecipitated with agarose conjugated anti-Jak2 antibody and then immunoblotted with anti-CaM or anti-Jak2 antibody. The insert is a representative immunoblot (CaM). The same blot was stripped and re-probed with antibodies for total Jak2 to ensure an equal loading of proteins (Jak2). (B) After stimulation of podocytes with 10 ng/ml of EGF for 10 min in the presence or absence of AG490 (50 μM) or AG1478 (20 μM), precleared cell lysates were immunoprecipitated with agarose conjugated anti-NHE-1 antibody and then immunoblotted with anti- calmodulin or anti-NHE-1 antibody. Values are mean±SEM from 3 independent experiments. ⁎Pb0.05 vs control, ‡Pb0.05 vs EGF.

in medium in which sodium was replaced by TMA. In addition, 5 μMof 5-(N-methyl-N-isobutyl) amiloride (MIA), an inhibitor of NHE-1 and NHE-2, attenuated EGF-induced proton efﬂux by nearly 60% (Fig. 3B). These ﬁndings suggest that EGF-induced increases in ECAR are due to NHE-1 or NHE-2 in podocytes.

3.5. Calmodulin inhibitors, phosphotyrosine inhibitors and Jak2 Fig. 4. Effects of CaM inhibitors and tyrosine kinase inhibitors on EGF-induced ECAR in inhibitors attenuate EGF-induced NHE-1 activity podocytes. Microphysiometry was used to measure ECAR in podocytes. (A) Podocytes were pretreated with CaM inhibitors for 30 min prior to stimulation with 10 ng/ml of NHE-1 has two CaM-binding domains that are critical for its EGF. (B) Podocytes were pretreated with AG1478 (20 μM) or AG490 (50 μM) for activation by many stimuli [41,42], whereas the role of CaM in the 30 min prior to stimulation with 10 ng/ml of EGF. Values are mean±SEM of peak values from 3 independent experiments performed in triplicate. ⁎Pb0.05; ⁎⁎Pb0.01 vs regulation of NHE-2 is much less certain [43]. Although elevations of drug alone samples. intracellular calcium increase the activity of NHE-2 [43], CaM has been 1178 S.D. Coaxum et al. / Biochimica et Biophysica Acta 1793 (2009) 1174–1181 shown to exert tonic inhibition on NHE-2 [34]. To determine whether 3.6. EGF increases formation of complexes of Jak2 and NHE-1 with CaM CaM is involved in EGF induced increases in ECAR, we analyzed the effects of a panel of CaM inhibitors on EGF-induced proton efflux in To further examine a role for Jak2 in EGF-induced signaling, we podocytes. The results in Fig. 4A demonstrate that W-7, fluphenazine, determined whether EGF stimulates the formation of signaling and ophiobolin A, each inhibited EGF-induced increases in ECAR by complexes between Jak2, NHE-1, and CaM. To explore this possibility, ≥60%. Because none of those agents reduced the basal levels of proton we performed co-immunoprecipitation experiments using cell lysates efflux in podocytes, the results are most consistent with EGF activation from podocytes pretreated with vehicle or with inhibitors of Jak2 or of NHE-1. EGFR tyrosine kinases. Fig. 5A shows that CaM was present in Jak2 Because previous studies from our laboratory demonstrated that immunoprecipitates, and that the amount of CaM present in these

Jak2 is important for NHE-1 activation by hypertonicity and by Gq- immunoprecipitates was doubled after EGF stimulation. Pretreatment coupled receptors [32,33], we analyzed the effects of a Jak2 inhibitor, of cells with a Jak2 inhibitor, AG-490 (50 μM for 30 min) signiﬁcantly AG490, on EGF-induced activation of NHE-1 in podocytes. AG490 decreased the amount of CaM in Jak2 immunoprecipitates, whereas (50 μM) inhibited EGF-induced increases in ECAR by N50% (Fig. 4B). pretreatment with an EGFR kinase inhibitor, AG1478 (20 μM for The EGFR tyrosine kinase inhibitor AG1478 (20 μM) also inhibited 30 min) did not have such effect. This result suggests that EGF- ECAR in podocytes that were stimulated with EGF by N95%. These induced Jak2 activity (but not EGFR kinase activity) is necessary for results support the involvement of Jak2 and the EGFR in the EGF- formation of the complex between Jak2 and CaM. In addition, Fig. 5B induced increases in ECAR. shows that there was a marked increase in the amount of CaM in NHE-

Fig. 6. Tyrosine phosphorylation of Jak2 and CaM in response to EGF treatment of podocytes. Co-immunoprecipitation experiments were performed as described under Materials and methods. (A) Podocytes were pretreated with AG-490 (50 μM), or with AG-1478 (20 μM), or vehicle for 30 min, and stimulated with 10 ng/ml of EGF for 5 min prior to cell lysates being immunoprecipitated with anti-phosphotyrosine/protein A-agarose, and then immunoblotted with polyclonal anti-EGFR antibody (A), or with polyclonal anti-Jak2 antibody (B). (C) To determine if CaM was tyrosine phosphorylated, podocytes were stimulated with 10 ng/ml of EGF for 5 min after pretreatment with AG-490 (50 μM), or with AG-1478 (20 μM) or vehicle for 30 min, immunoprecipitated with monoclonal anti-phosphotyrosine (PY) antibody conjugated with protein A-agarose and then immunoblotted with a monoclonal anti-CaM antibody. The same blots were stripped and re-probed with polyclonal PY antibody to conﬁrm that CaM is indeed tyrosine phosphorylated in response to stimulation with EGF (not shown). Values are mean±SEM from 5 independent experiments. ⁎Pb0.05 vs control, ‡Pb0.05 vs EGF. S.D. Coaxum et al. / Biochimica et Biophysica Acta 1793 (2009) 1174–1181 1179

1 immunoprecipitates after treatment with EGF. In contrast, there was not an increased formation of complexes between Jak2 and NHE-1 in podocytes after treatment with EGF (not shown). Pretreatment of cells with a Jak2 inhibitor, AG490 (50 μM) or EGFR kinase inhibitor, AG1478 (20 μM) decreased the amount of CaM in NHE-1 immunoprecipitates. The latter result suggests that both EGFR kinase activity and Jak2 activity are required to induce formation of a complex between CaM and NHE-1.

3.7. EGF induces tyrosine phosphorylation of Jak and CaM

In order to examine further the signaling mechanisms involved in the activation of NHE-1 by EGF, we next considered that EGF could stimulate tyrosine phosphorylation of CaM. The data presented in Fig. 6 (panel A) demonstrate that EGF increased the amount of EGFR in phosphotyrosine immunoprecipitates, and that this effect is unchanged in the presence of Jak2 inhibitor, but is completely abolished after pretreatment with AG1478. This result demonstrates that AG1478 effectively inhibits intrinsic EGFR tyrosine kinase activity in podocytes. Fig. 6 (panel B) shows that EGF induces Fig. 7. Schematic representation of two converging pathways of activation of NHE-1 in tyrosine phosphorylation of Jak2, which is inhibited by pretreatment podocytes by EGF. P-CaM indicates tyrosine phosphorylated CaM. All other abbrevia- with AG-490, but not with AG-1478. These results provide strong tions are as described in the text. evidence that EGF induces tyrosine phosphorylation of EGFR and Jak2 via auto-phosphorylation of these kinases, and also demonstrate that AG-490 and AG-1478 were effective under our experi- tyrosine phosphorylated through a pathway that is inhibited by mental conditions. The results also suggest that EGFR kinase activity AG490, and (3) CaM is a bona fide substrate for Jak2 [31]. is not required for Jak2 activation by EGF. Fig. 6 (panel C) demonstrates The evidence implicating CaM in the increase in proton efflux is that EGF increases the amount of CaM in phosphotyrosine immuno- that (1) a panel of CaM inhibitors greatly attenuates the increased precipitates and that this effect can be significantly decreased by proton efflux induced by EGF, (2) CaM is tyrosine phosphorylated in pretreatment of cells with AG-490, but not with AG-1478, suggesting response to EGF, and (3) CaM is induced to form complexes with Jak2 that tyrosine phosphorylation of CaM is induced by Jak2, and does not and NHE-1 in response to EGF. The evidence that the proton efflux is require EGFR kinase activity. In that regard, we demonstrated mediated by NHE-1 is that it is (1) dependent upon extracellular previously that CaM is a bona fide substrate for Jak2 [31]. sodium, (2) inhibited by MIA, (3) dependent upon CaM activity, and (4) associated with increased binding of CaM to NHE-1. 4. Discussion The precise mechanism through which Jak2 activates NHE-1 has not been fully elucidated. We propose that Jak2 tyrosine phosphor- What is new about this work is that we have demonstrated that ylates CaM, thereby increasing its affinity for NHE-1. This would result EGF activates NHE-1 through the intermediary actions of Jak2 and in increased binding of CaM to NHE-1. A number of kinases have been CaM in renal podocytes. The work expands recent studies demon- shown to phosphorylate CaM on serine, threonine and tyrosine strating that hypertonicity and Gq-coupled receptors activate NHE-1 residues [44,45], and to alter the activity of CaM with reference to in several cell types through a pathway involving sequential specific CaM targets [46]. In that regard, our group has recently phosphorylation and activation of Jak2, tyrosine phosphorylation of demonstrated that CaM is directly tyrosine phosphorylated by CaM, CaM binding to NHE-1, and activation of NHE-1. The current purified Jak2 [31]. Thus, Jak2 almost certainly phosphorylates CaM work is significant in that we have (1) demonstrated that a on one or both of the tyrosine residues within the CaM sequence, Tyr- prototypical receptor tyrosine kinase (the EGFR) utilizes this pathway 99 and Tyr-138. Based on the crystal structure of CaM, Tyr-99 is the and a second pathway, both of which are required for full activation of more likely target for phosphorylation in that Tyr-99 is located within NHE-1; (2) refined the previously identified pathway as follows: the third Ca2+-binding domain, and is somewhat more exposed than EGF→EGFR→Jak2 activation (independent of EGFR tyrosine kinase is Tyr-138 [45]. However, Jak2-induced tyrosine phosphorylation of activity)→tyrosine phosphorylation of CaM→CaM binding to NHE- CaM appears to be critical or necessary, but not sufficient to fully 1 → activation of NHE-1; (3) characterized a second activation activate NHE-1, because EGFR tyrosine kinase activity also is required. pathway as follows: EGF→EGFR→EGFR kinase activation→associa- Indeed, the effectiveness of AG1478 to block NHE-1 activation tion of CaM to NHE-1 (independent of Jak2)→activation of NHE-1 suggests that EGFR tyrosine kinase activity also is essential for CaM (Fig. 7). (4) We also have identified mRNAs for various isotypes of to bind to NHE-1 and to activate it. It should be noted that we have not plasma membrane NHEs, and for EGFR-related subunits, in renal formally tested the idea that CaM binding to NHE-1 induces a podocytes. Because podocytes have been implicated as playing key conformational change that results in activation of NHE-1. However, roles in the initial stages of various glomerular diseases, this new this idea is intuitively pleasing, and has been supported by experi- information may have relevance to the processes that link podocyte mental evidence in the form of mutation studies by [43], and by dysfunction to progressive renal diseases. solution phase spectroscopy studies of the interaction between CaM The evidence implicating Jak2 in the increase in proton efflux is and the large regulatory intracellular carboxyl terminus of NHE-1 by that (1) Jak2 is activated as demonstrated by its tyrosine phosphor- Fliegel's group [41]. ylation in response to EGF, (2) AG490 blocks the increased proton It is important to elaborate on our findings that the EGFR kinase efflux induced by EGF, and (3) Jak2 forms a complex with CaM in inhibitor AG1478 did not decrease the amount of Jak2 and CaM in response to EGF. Although our work does not prove definitively that phosphotyrosine immunoprecipitates (Fig. 5B and C, respectively), tyrosine phosphorylation of Jak2 is required for activation of NHE-1 by which suggests that there is another factor that allows EGF to regulate EGF, this seems likely in that (1) EGF does not increase intracellular tyrosine phosphorylation of CaM independent of EGFR kinase activity. calcium levels under our conditions (data not shown), (2) CaM is This finding is supported by previous reports that suggest that some 1180 S.D. Coaxum et al. / Biochimica et Biophysica Acta 1793 (2009) 1174–1181

EGF-mediated signals such as the JAK/STAT pathway are independent Affairs (Merits Awards and a REAP award to JRR and MNG), the of EGFR kinase activity [47,48]. Two groups demonstrated that National Institutes of Health (DK52448 and GM63909 to JRR, and AG1478-independent effects of EGF might be mediated by ErbB2 DK52448-S2 to SDC), the American Heart Association (GIA (Neu/HER2), possibly through oligomerization with ErbB1/EGFR 0655445U to MNG), and a laboratory endowment jointly supported [47,48]. It is unlikely that this mechanism can account for our findings by the M.U.S.C. Division of Nephrology and Dialysis Clinics, Inc. (JRR). in that we detected little to no Neu/HER2 mRNA in differentiated The work also was supported by VA shared equipment grants podocytes (Fig. 2A). (confocal microscope and microphysiometer). An alternative explanation for the dual Jak2- and EGFR tyrosine kinase-dependent pathways of activation of NHE-1 is that both EGFR References and Jak2 could tyrosine phosphorylate CaM. This idea is reasonable because the EGFR has been shown to phosphorylate CaM on Tyr-99 [1] G. Nowak, R.G. Schnellmann, Integrative effects of EGF on metabolism and and/or Tyr-138 in other cell systems [44,49]. Indeed, the EGFR proliferation in renal proximal tubular cells, Am. J. Physiol. 269 (1995) – possesses a juxtamembrane CaM-binding motif at residues 624–639, C1317 C1325. [2] R.P. Schaudies, D. Nonclercq, L. Nelson, G. Toubeau, J. Zanen, J.A. Heuson-Stiennon, which Martin-Nieto and Villalobo demonstrated could bind to CaM in G. Laurent, Endogenous EGF as a potential renotrophic factor in ischemia-induced a calcium-dependent manner, with an affinity of ≈400 nM [50]. acute renal failure, Am. J. Physiol. 265 (1993) F425–F434. However, it seems unlikely that the EGFR directly phosphorylates CaM [3] F. Terzi, M. Burtin, M. Hekmati, P. Federici, G. Grimber, P. Briand, G. fi Friedlander, Targeted expression of a dominant-negative EGF-R in the kidney in podocytes in that the Jak2 inhibitor, AG490, signi cantly suppresses reduces tubulo-interstitial lesions after renal injury, J. Clin. Invest. 106 (2000) EGF-induced tyrosine phosphorylation of CaM, whereas AG1478 has 225–234. no significant effect (Fig. 6B). [4] H. Sakurai, T. Tsukamoto, C.A. Kjelsberg, L.G. Cantley, S.K. Nigam, EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by Because AG1478 attenuates ECAR more than CaM or Jak2 embryonic kidney, Am. J. Physiol. 273 (1997) F463–F472. inhibitors, it appears that the receptor tyrosine kinase activity of [5] C. Fleck, J. 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