Arachidonic acid induces ERK activation via Src SH2 domain association with the epidermal growth factor receptor

original article

http://www.kidney-international.org & 2006 International Society of Nephrology

Arachidonic acid induces ERK activation via Src SH2 domain association with the epidermal growth factor receptor LD Alexander1, Y Ding2, S Alagarsamy2, X-L Cui2 and JG Douglas2 1

Department of Natural Sciences, The University of Michigan-Dearborn, Dearborn, Michigan, USA and 2Department of Medicine, Division of Nephrology and Hypertension, Case Western Reserve University School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio, USA

Within the kidney, angiotensin II type 2 (AT2) receptor mediates phospholipase A2 (PLA2) activation, arachidonic acid release, epidermal growth factor (EGF) receptor transactivation, and mitogen-activated protein kinase activation. Arachidonic acid mimics this transactivation by an undetermined mechanism. The role of c-Src in mediating angiotensin II and arachidonic acid signaling was determined by employing immunocomplex kinase assay, Western blotting analysis, and protein immunoblotting on co-precipitated EGF receptor (EGFR) proteins and agarose conjugates of glutathione S-transferase fusion proteins containing the c-Src homology 2 (SH2) and SH3 domains. Angiotensin II induced extracellular signal-regulated kinase (ERK) activation in primary cultures of rabbit proximal tubule cells via the activation of c-Src and association of the EGFR with the c-Src SH2 domain, effects that were mimicked by arachidonic acid and its inactive analogue eicosatetraynoic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphate, AT2 receptor by PD123319, Src family kinases by, 1-(tert-butyl)-3-(4-chlorophenyl)-4aminopyrazolo[3,4-d] pyrimidine (PP2) and c-Src by overexpression of a dominant-negative mutant of c-Src abrogated these effects. However, inhibitors of arachidonic acid metabolic pathways did not block these effects. The present work provides a new and novel paradigm for transactivation of a kinase receptor linked to a fatty acid, which may apply to activation of a variety of phospholipases and accompanying arachidonic acid release. Kidney International (2006) 69, 1823–1832. doi:10.1038/sj.ki.5000363; published online 5 April 2006 KEYWORDS: arachidonic acid; angiotensin II; c-Src; EGF receptor transactivation; mitogen activated-protein (MAP) kinase

Correspondence: LD Alexander, Department of Natural Sciences, The University of Michigan-Dearborn, 4901 Evergreen Road, Dearborn, Michigan 48128, USA. E-mail: [email protected] Received 21 November 2003; revised 16 August 2005; accepted 15 September 2005; published online 5 April 2006 Kidney International (2006) 69, 1823–1832

Molecular biological observations validate the existence of two major angiotensin II receptor subtypes (type 1 (AT1) and type 2 (AT2)). These receptor subtypes are pharmacologically distinct and share only approximately 30% sequence homology. While the AT1 receptor has been considered to subserve many of the biological functions linked to angiotensin II, emerging evidence indicates that the adult AT2 receptor may also mediate significant biological responses as well. Renal proximal tubule epithelial cells represent an unusual site of angiotensin II action, as both angiotensin II AT1 (basolateral) and AT2 (apical) receptors are present. This rabbit AT2 receptor is novel with respect to pharmacological characteristics, in that pharmacologically it is insensitive to losartan and candesartan, but sensitive to PD123319,1 and its unique signaling is linked to arachidonic acid release upon activation.2–4 In contrast to the AT2 receptor expressed in PC12 cells, which inhibits extracellular signal-regulated kinase (ERK) activation, a member of the mitogen-activated protein (MAP) kinases, the rabbit AT2 receptor activates ERK through Shc/Grb2/Sos and p21ras.5,6 Src proteins consist of at least 14 related alternatively spliced gene products, which are expressed in a tissue-specific manner.7 A growing body of data indicates that the Src family of protein tyrosine kinases functionally interacts with a variety of non-receptor and receptor protein tyrosine kinases, particularly the epidermal growth factor (EGF) receptor (EGFR).8–11 From these studies, it is generally agreed that the association of Src (through Src homology 2 (SH2) domain) with the EGFR results in the mutual stimulation of Src catalytic activity, and enhanced phosphorylation of the receptors’ downstream targets. In most cell types, the tyrosine phosphorylation of the EGFR, in response to angiotensin II, was observed to be mediated by the angiotensin II type 1 (AT1) receptor.8,9,12 However, in rabbit proximal tubule cells, the effects of angiotensin II on EGFR phosphorylation appear to be mediated by AT2 receptor,6,13 and arachidonic acid appears to mimic and mediate angiotensin II-induced signal transduction. Although mechanisms linking the AT1 receptor to the tyrosine kinase pathway exist, unresolved questions 1823

RESULTS Angiotensin II-induced Src tyrosine phosphorylation

We recently reported that arachidonic acid induces c-Src activation in a time- and dose-dependent manner in rabbit proximal tubule cells.14 Considering that arachidonic acid mimics and mediates the biological functions of angiotensin II by the AT2-receptor subtype in these same cells,5,6 we determined whether angiotensin II activated c-Src. As shown in Figure 1a, angiotensin II (1 mmol/l) significantly increased c-Src phosphorylation in a time-dependent manner. Maximum effect was observed at 5 min and the response declined at 15–30 min, but was still significant, and then returned to basal levels by 60 min. c-Src phosphorylation by angiotensin II was concentration dependent, in that it significantly increased at 10
7 mol/l angiotensin II for 5 min and remained similar and higher at 10
6 and 10
5 mol/l of angiotensin II (Figure 1b). Therefore, subsequent assays of angiotensin IIinduced c-Src phosphorylation utilized 10
6 mol/l angiotensin II stimulation for 5 min. These results were confirmed with an in vitro kinase assay. A significant increase (2517 36% compared to controls) in phosphorylation of an Srcspecific substrate peptide (activity) by arachidonic acid, in a manner similar to angiotensin II (263748%), was observed, which paralleled the increase in c-Src phosphorylation (Figure 2). In addition, the Src family kinase inhibitor PP2 significantly reduced c-Src phosphorylation by arachidonic acid and angiotensin II in a dose-dependent manner (Figure 3a and b, respectively). Similar inhibition was demonstrated by transfection of proximal tubule cells with an Src dominant-negative mutant cDNA (Figure 2). In addition, PD123319, a selective AT2-receptor blocker, also 1824

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persist as to the mechanism(s) by which the AT2 receptor and arachidonic acid, a fatty acid, are linked to the tyrosine kinase pathway. In the present study, we examined whether angiotensin II stimulates c-Src and the role of c-Src in mediating angiotensin II-induced EGFR transactivation and ERK activation. In addition, we evaluated whether cytosolic phospholipase A2 (cPLA2) and arachidonic acid might act as mediators of angiotensin II signaling in these proximal tubular cells. Herein, we demonstrate for the first time that angiotensin II induces ERK phosphorylation and activation in primary cultures of rabbit proximal tubule cells via an AT2-induced activated c-Src and its SH2 domain association with the EGFR. In addition, our data demonstrate that arachidonic acid mimics and mediates the effects of angiotensin II, independent of eicosanoid biosynthesis. To our knowledge, the ability of arachidonic acid to activate c-Src and induce binding of the EGFR to a glutathione S-transferase (GST) fusion protein containing the c-Src SH2 domain to modulate EGFR transactivation and ERK activation is entirely novel and has not been reported previously. Thus, this may serve as a new signaling paradigm, since arachidonic acid and other fatty acids are linked to the tyrosine kinase pathway, an effect that may extend to other cell types.

LD Alexander et al.: Arachidonic acid induces c-Src activation

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original article

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Figure 1 | Angiotensin II-induces c-Src activation in a time- and dose-dependent manner in rabbit proximal tubule cells. (a) Serum-starved proximal tubule epithelial cells were exposed to either angiotensin II for the indicated times or (b) various concentrations of angiotensin II (0–10 mmol/l) for 5 min. The cells were then lysed and equal amounts of proteins were used for Western blotting. c-Src was evaluated by Western blot analysis using an anti-phosphorylated c-Src (pY418)-specific antibody (upper panel). The same blots were stripped and reprobed with control anti-c-Src antibody, showing equal loading and confirming the signal detected with the phospho-antibody (lower panels). The autoradiograph shows a representative Western blot. Bar graphs represent intensities of phospho-c-Src quantified by scanning densitometry of blots and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple-comparison test. Statistical significance: ***Po0.001 versus unstimulated control cells.

abrogated the phosphorylation of c-Src. Losartan (Lor) and candesartan (Cand), two selective AT1-receptor blockers, did not alter angiotensin II-elicited actions (Figure 4a). Pretreatment of proximal tubular cells with 17-octadecynoic acid (10 mmol/l), a potent inhibitor of both o-hydroxylase and epoxygenase enzyme activity, indomethacin (50 mmol/l), a cyclooxygenase inhibitor, or nordihydroguaiaretic acid (10 mmol/l), a lipoxygenase inhibitor, did not affect arachidonic acid and angiotensin II-induced c-Src activation (data not shown). By analogy, angiotensin II-induced c-Src activation was totally abrogated by the cPLA2 inhibitors methyl arachidonyl fluorophosphate (MAFP) (10474%, Po0.001) and mepacrine (107711%, Po0.001), whereas haloenol lactone suicide substrate, a potent irreversible inhibitor of Ca2 þ -independent phospholipase A2 (PLA2), did not interfere with angiotensin II-induced c-Src activation (Figure 4b). Collectively, these results demonstrate that Kidney International (2006) 69, 1823–1832

original article

LD Alexander et al.: Arachidonic acid induces c-Src activation

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Figure 2 | Arachidonic acid and angiotensin II increases c-Src kinase activity. Rabbit proximal tubular epithelial cells were grown to confluency and serum starved for 48 h. The cells were pretreated with PP2 (10 mmol/l) or transfected with control plasmid cDNA (empty vector) or dominant-negative c-Src K298M (c-Src DN), and were made quiescent with serum-free medium for 24 h. They were then stimulated with arachidonic acid (15 mmol/l) or angiotensin II (1 mmol/l) for 5 min. Activation of c-Src kinase was detected as described in Materials and methods. Values are represented as mean7s.e.m. of three independent experiments and are expressed as percent relative to control with unstimulated as 100%. Statistical comparisons were made with Student’s t-test or a one-way analysis of variance (ANOVA), followed by Newman–Keuls multiple-comparison test. Statistical significance: Asterisks indicate a significant effect of arachidonic acid or angiotensin II treatment compared with the non-stimulated-empty vector control cells (***Po0.001). þ þ Po0.01, þþþ Po0.001 indicate significant effect of PP2 or c-Src DN construct compared with the arachidonic acid- and angiotensin II-stimulated cells.

angiotensin II-induced c-Src activation in kidney epithelium occurs via AT2 receptor-dependent signaling pathways and that angiotensin II evokes arachidonic acid release by stimulating cPLA2, but not independent phospholipase A2 in rabbit proximal tubule cells. Moreover, arachidonic acid-induced c-Src activation appears to be direct and independent of eicosanoid biosynthesis. Angiotensin II and arachidonic acid activate extracellular signal-regulated kinase via c-Src-dependent phosphorylation of the EGFR

Earlier studies, by this laboratory, demonstrated that angiotensin II and arachidonic acid stimulated phosphorylation of EGFR in cultured proximal tubule cells.13 Likewise, EGFR transactivation appears to be a mediator of angiotensin II and arachidonic acid signaling, leading to ERK activation. To examine the role of c-Src activation in EGFR transactivation and ERK activation by angiotensin II and arachidonic acid, cells were pretreated either with the Src family kinase inhibitor PP2 or transiently transfected with a dominant-negative mutant of c-Src. Blockade of Src family kinases using PP2 significantly decreased arachidonic acid- and angiotensin II-induced ERK activation in a dosedependent manner (Figure 5a and b, respectively). PP2 treatment, however, did not affect EGF-induced EGFR tyrosine phosphorylation and ERK activation (Figure 6a and b, respectively). Similarly, transfection of proximal tubule cells with dominant-negative c-Src did not affect the ability of EGF to stimulate EGFR tyrosine phosphorylation (Figure 7b) Kidney International (2006) 69, 1823–1832

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Figure 3 | Effects of Src family kinase inhibitor PP2 on arachidonic acid- and angiotensin II–c-Src activation. Rabbit proximal tubular epithelial cells were grown to confluency and serum starved for 48 h. Quiescent cells were incubated with or without PP2 (1, 10, and 20 mmol/l) for 30 min, followed by exposure to (a) arachidonic acid (15 mmol/l) or (b) angiotensin II (1 mmol/l) for 5 min. The cells were then lysed and equal amounts of proteins were resolved by 10% SDS-PAGE and immunoblotted with an anti-phospho-c-Src polyclonal antibody (p-Src (pY418), upper blots). The same blots were stripped and reprobed with an anti-c-Src antibody to demonstrate equal protein loading (lower blots). The autoradiograph shows a representative Western blot. (a, b) Densitometric analysis of the experiments is reported. Bar graphs represent intensities of phospho-c-Src quantified by scanning densitometry of blots and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple-comparison test. Statistical significance: Asterisks indicate a significant effect of PP2 treatment compared with the arachidonic acid- and angiotensin II-stimulated cells (**Po0.01; ***Po0.001). þ þ Po0.01; þþþ Po0.001 indicate significant effects of arachidonic acid and angiotensin II treatment compared with the non-stimulated (DMSO-treated) control cells.

and ERK activation (Figure 7c), but significantly reduced angiotensin II- and arachidonic acid-induced c-Src activation, EGFR tyrosine phosphorylation and ERK activation (Figure 7a–c, respectively). By analogy, pretreatment with the selective EGFR antagonist AG1478 greatly reduced angiotensin II-, arachidonic acid-, and EGF-induced EGFR tyrosine phosphorylation (Figure 8a), but had no effect on the capacity of angiotensin II and arachidonic acid to induce the activation of c-Src (Figure 8b). In addition, EGF treatment appeared to have no effect on c-Src activation (Figures 7a and 8b, respectively). Moreover, among the members of the Src family kinases, only c-Src was specifically coimmunoprecipitated with the EGFR (Figure 9a and b). In addition, arachidonic acid-induced c-Src association with the EGFR 1825

original article

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Figure 5 | Effects of Src family kinase inhibitor PP2 on arachidonic acid- and angiotensin II-induced ERK phosphorylation. Rabbit proximal tubular epithelial cells were grown to confluency and serum starved for 48 h. Cells were pretreated with PP2 (1, 10 or 20 mmol/l) for 30 min, followed by (a) arachidonic acid (15 mmol/l) or (b) angiotensin II (1 mmol/l) for 5 min. The cells were then lysed and total cellular lysates (20 mg) were immunoblotted with antibodies to ERK 1/ 2 phospho-threonine 202/tyrosine204 (pERK 1/2; upper panel). The same blots were stripped and reprobed with a control anti-ERK1/2 antibody showing equal loading (lower panel). The autoradiograph shows a representative Western blot. Bar graphs represents the intensities of phospho-ERK 1/2 quantified by scanning densitometry of blots and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple-comparison test. Statistical significance: ***Po0.001 versus stimulated cells after PP2 treatment.

Figure 4 | Effects of angiotensin II-receptor antagonist on angiotensin II-induced c-Src phosphorylation. Rabbit proximal tubular epithelial cells were grown to confluency and serum starved for 48 h. (a) Quiescent cells were incubated with or without losartan (Lor,10 mmol/l), candesartan (Cand,10 mmol/l) or PD123319 (PD,10 mmol/l) for 60 min, followed by exposure to angiotensin II (1 mmol/l) for 5 min or (b) incubated with or without 10 mmol/l MAFP or 25 mmol/l haloenol lactone suicide substrate (HELSS) for 30 min or 500 mmol/l mepacrine for 5 min, and then exposed to 1 mmol/l angiotensin II for 5 min. The cells were then harvested and equal amounts of proteins were resolved by 10% SDS-PAGE and immunoblotted with an anti-phospho-c-Src polyclonal antibody (p-Src (pY418), upper blots). The same blots were stripped and reprobed with an anti-c-Src antibody to demonstrate equal protein loading (lower blots). The autoradiograph shows a representative Western blot. Bar graphs represent intensities of phospho-c-Src quantified by scanning densitometry of blots, and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of at least three independent experiments. Statistical significance: ***Po0.001 versus nonstimulated control with the same group; þ þ þ Po0.001, þ þ Po0.01; Lor, Cand, PD, MAFP, HELSS or mepacrine (MEP) versus angiotensin II-stimulated cells.

was blocked by pretreatment of the cells with the Src family kinase inhibitor PP2 (Figure 9a) and by transient expression of dominant-negative c-Src (Figure 9b). Thus, we have demonstrated for the first time that, among the members of the Src family kinases, only c-Src specifically coimmunoprecipitate with the EGFR. Collectively, these data suggest that c-Src is involved in angiotensin II- and arachidonic acidinduced transactivation of the EGFR and ERK activation, and

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LD Alexander et al.: Arachidonic acid induces c-Src activation

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Figure 6 | Src family kinase inhibitor has no effect on EGFinduced EGFR tyrosine phosphorylation and ERK activation. Rabbit proximal tubular epithelial cells were grown to confluency and serum starved for 48 h. Cells were pretreated with PP2 (10 mmol/l) for 30 min, followed by EGF (10 ng/ml) for 5 min. The cells were then lysed and total cellular lysates were (a) immunoprecipitated (0.5 mg protein) with an anti-EGFR (1005) rabbit polyclonal antibody followed by blotting with anti-phosphotyrosine (PY20) antibody (upper panels), or (b) immunoblotted (20 mg) with antibodies to ERK 1/2 phospho-threonine 202/tyrosine204 (pERK 1/2; upper panel). The same blots were stripped and reprobed with control anti-EGFR or anti-ERK1/2 antibodies showing equal loading (lower panels). The autoradiograph shows a representative Western blot. Bar graphs represent intensities of phospho-EGFR or phospho-ERK 1/2 quantified by scanning densitometry of blots, and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a Student’s t-test. Statistical significance: NS versus stimulated cells after PP2 treatment. Kidney International (2006) 69, 1823–1832

original article

LD Alexander et al.: Arachidonic acid induces c-Src activation

a EGF (10 ng/ml):

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Figure 7 | Angiotensin II and arachidonic acid activate ERK via c-Src-dependent phosphorylation of the EGFR in rabbit proximal tubule cells. Rabbit proximal tubular epithelial cells were grown to 80% confluency, transfected with empty vector (pCMV5) or dominant-negative c-Src K298M (c-Src DN), and subsequently made quiescent with serum-free medium for 24 h. Cells were then incubated either in the presence or absence of angiotensin II (1 mmol/l), arachidonic acid (15 mmol/l) or EGF (10 ng/ml) for 5 min. The cells were then lysed and equal amounts of proteins were subjected to immunoblot analysis with polyclonal antibodies that specifically recognize the phosphorylated forms of (a) c-Src or (c) ERK 1/2, or (b) were immunoprecipitated with an anti-EGFR (1005) rabbit polyclonal antibody followed by blotting with anti-phosphotyrosine (PY20) antibody (upper panels). The same blots were stripped and reprobed with control anti-c-Src, anti-ERK 1/2, or anti-EGFR antibodies showing equal loading (lower panels). The autoradiographs represent the results of a single experiment, which was carried out at least three times.

Figure 8 | Effects of EGFR kinase inhibitor AG1478 on angiotensin II- and arachidonic acid-induced EGFR tyrosine phosphorylation and c-Src activation in rabbit proximal tubule cells. Rabbit proximal tubular epithelial cells were grown to confluency and serum starved for 48 h. Cells were pretreated with AG1478 (500 nmol/l) for 60 min, followed by angiotensin II (1 mmol/l), arachidonic acid (15 mmol/l) or EGF (10 ng/ml) for 5 min. The cells were then lysed and (0.5 mg protein) were (a) immunoprecipitated with an anti-EGFR (1005) rabbit polyclonal antibody followed by blotting with anti-phosphotyrosine (PY20) antibody, or equal amounts of proteins were used for Western blotting. The blots were probed with (b) rabbit polyclonal anti-phosphorylated c-Src (pY418) (upper panels). The same blots were stripped and reprobed with control anti-EGFR or ant-c-Src antibodies showing equal loading (lower panels). The autoradiograph represents the results of a single experiment, which was carried out at least three times.

a Arachidonic acid (min): PP2 (10
mol/l): IP: c-Src IB: EGFR IP: c-Src IB: c-Src

b Arachidonic acid (min): is consistent with c-Src being upstream of EGFR tyrosine phosphorylation and ERK activation. Arachidonic acid induces binding of EGFR with the c-Src homology 2 domain GST fusion protein

We evaluated whether there was an association of the EGFR with the c-SH2 domain. Here cell lysates were immunoprecipitated with a c-Src GST–SH2 fusion protein and subsequently immunoblotted with monoclonal anti-EGFR antibodies. As shown in Figure 10a, arachidonic acid induced binding of EGFR with the c-Src SH2 domain fusion protein in a time-dependent manner that was significant at 5 min, maximal at 15 min, and returned to basal levels by 60 min. This effect was significant at 5 mmol/l and reached a plateau by 15 mmol/l (Figure 10b). Angiotensin II stimulated an association of c-Src SH2 domain with the EGFR in a manner similar to arachidonic acid (Figure 10c and d). In addition, PP2 inhibited arachidonic acid- and angiotensin II-induced association of EGFR with the c-Src SH2 domain GST fusion protein by 6475% (Po0.01) and 6072% (Po0.001), respectively, while having no effect on EGF-induced association of EGFR with the c-Src SH2 domain of GST fusion Kidney International (2006) 69, 1823–1832

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Figure 9 | Arachidonic acid-induced EGFR association with c-Src is dependent of c-Src. Serum-starved proximal tubular cells were either pretreated with (a) PP2 (10 mmol/l) or (b) transfected with control plasmid or dominant-negative c-Src K298M (c-Src DN constructs) serum restricted for 48 h, followed by stimulation with arachidonic acid (15 mmol/l) for 5 min. The cells were then lysed and total cellular lysates (0.5 mg protein) were immunoprecipitated with anti-c-Src monoclonal antibody, followed by blotting with anti-EGFR polyclonal antibody (upper panels). The membrane was stripped and reprobed with an anti-c-Src monoclonal antibody (bottom panels). The autoradiographs represent the results of a single experiment, which was carried out at least three times.

protein (Figure 11a). Similarly, overexpression of dominantnegative c-Src totally abrogated arachidonic acid- and angiotensin II-induced association of EGFR with the c-Src SH2 domain GST fusion protein, while having no effect on EGFinduced association of EGFR with the c-Src SH2 domain GST fusion protein (Figure 11b). Collectively, these data demonstrate that in early passaged kidney epithelial cells, arachidonic acid- and angiotensin II-induced transactivation 1827

original article

LD Alexander et al.: Arachidonic acid induces c-Src activation

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Figure 10 | Arachidonic acid- and angiotensin II-induced time- and dose-dependent association of Src–SH2 domain with the EGFR. Serum-starved proximal tubular cells were (a) treated either without or with 15 mmol/l arachidonic acid or (c) 1 mmol/l angiotensin II for the indicated times or incubated with various concentrations of (b) arachidonic acid or (d) angiotensin II for 5 min at 371C. Cells were lysed and immunoprecipitated (IP) with GST-Src SH2-Sepharose as described in Materials and methods. Precipitated proteins were resolved by SDS-PAGE and immunoblotted (IB) with polyclonal anti-EGFR antibody (upper panels). The membrane was stripped and reprobed with an anti-c-Src monoclonal antibody (bottom panels). Comparable amounts of proteins were detected in immunoprecipitates of all the above experiments. The autoradiographs represent the results of a single experiment, which was carried out at least three times. Bar graphs represent intensities of associated EGFR quantified by scanning densitometry of blots and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple-comparison test. Statistical significance: ***Po0.001, **Po0.1, *Po0.05 versus unstimulated control cells.

of the EGFR occur via inducing the association of EGFR with the SH2 domain of the Src protein, and that the tyrosine phosphorylation of c-Src is involved. Arachidonic acid induces association of EGFR with the c-Src homology 2 domain GST fusion protein independent of eicosanoid biosynthesis

We have previously demonstrated that unsaturated longchain free fatty acids such as linoleic and linolenic acid potently activated members of the MAP-kinase superfamily15 in rabbit proximal tubule cells. As shown in Figure 12, stimulation of proximal tubule cells with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolized analog of arachidonic acid, which blocks arachidonic acid oxidization by acting as a false substrate,16,17 induced association of the EGFR with the c-Src SH2 domain GST fusion protein by 225723% (Po0.001), as compared with control. Two polyunsaturated fatty acids, linoleic acid (C18:2), and linolenic acid (C18:3), also significantly increased association of the EGFR with the c-Src SH2 domain GST fusion protein by 204717% and 203723%, respectively, as compared to control. By contrast, neither the monounsaturated fatty acid palmitoleic acid (C16:1) nor the two saturated fatty acids, stearic (C18), and arachidic acid (C20), had any effects on the 1828

association of the EGFR with the c-Src SH2 domain GST fusion protein. Again, the action of arachidonic acid was not affected by specific inhibitors of cyclooxygenase-, lipoxygenase-, and cytochrome P450-dependent pathways (data not shown), which is consistent with the notion that the effects of arachidonic acid or ETYA on the association of the EGFR with the c-Src SH2 domain GST fusion protein are specific effects and are therefore not dependent on an arachidonic acid metabolite. Specificity of binding of the EGFR with the c-Src SH2 domain versus SH3 domain was demonstrated, since arachidonic acid did not induce association of the EGFR with the c-Src SH3 domain GST fusion protein, an effect that was similar to angiotensin II (Figure 13). DISCUSSION

In prior reports, we demonstrated that angiotensin II and arachidonic acid rapidly activate a plethora of kinases, including the EGFR tyrosine kinase, p21ras, and members of the MAP kinase superfamily, particularly ERK, c-Jun NH2terminal kinase, and p38MAPK, in rabbit proximal tubule cells.6,13,15,18 However, the molecules involved in the signaling are not yet fully characterized. This report describes the mechanism of transmembrane signaling that is involved in Kidney International (2006) 69, 1823–1832

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Figure 11 | Association of the EGFR with Src SH2 domain is dependent on c-Src. Serum-starved proximal tubular cells were either pretreated with (a) PP2 (10 mmol/l) or (b) they were transfected with control plasmid or dominant negative c-Src K298M (c-Src DN constructs, serum restricted for 48 h. Cells were then incubated either in the presence or absence of arachidonic acid (15 mmol/l), angiotensin II (1 mmol/l), or EGF (10 ng/ml) for 5 min. Cells were lysed and immunoprecipitated (IP) with GST-Src SH2-Sepharose as described in Materials and methods. Precipitated proteins were resolved by SDS-PAGE and immunoblotted (IB) with polyclonal anti-EGFR antibody (upper panels). The membrane was stripped and reprobed with an anti-c-Src monoclonal antibody (bottom panels). Comparable amounts of proteins were detected in immunoprecipitates of all the above experiments. The autoradiographs represent the results of a single experiment, which was carried out at least three times. Bar graphs represents intensities of associated EGFR quantified by scanning densitometry of blots and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test. Statistical significance: þ þ þ Po0.001 indicates significant effect of PP2 or c-Src DN construct compared with the arachidonic acid-, angiotensin II-, and EGF-stimulated cells versus unstimulated control cells.

angiotensin II- and arachidonic acid-induced EGFR tyrosine phosphorylation and ERK activation. We report for the first time that (1) arachidonic acid itself, and not its metabolic metabolites, is the direct effector of c-Src activation; (2) arachidonic acid, ETYA and other fatty acids transactivate the EGFR via inducing association of SH2 domain with the EGFR; (3) angiotensin II- and arachidonic acid-induced c-Src association with the EGFR appears to be specific for c-Src SH2 domain versus SH3 domain and (4) c-src-mediated EGFR transactivation appears to be a critical component Kidney International (2006) 69, 1823–1832

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LD Alexander et al.: Arachidonic acid induces c-Src activation

Figure 12 | Arachidonic acid-induced association of Src–SH2 domain with the EGFR is fatty acid dependent, but eicosanoid independent. Serum-starved proximal tubular cells were exposed to equal molar concentrations (15 mmol/l) of arachidonic acid (C20:4), ETYA (C20:4), linoleic acid (C18:2), linolenic acid (C18:3), palmitoleic acid (C16:1), stearic acid (C18), or arachidic acid (C20) for 5 min at 371C. The numbers in parentheses indicate the carbon chain length followed by the number of double bonds. Cells were lysed and immunoprecipitated (IP) with GST-Src SH2-Sepharose as described in Materials and methods. Precipitated proteins were resolved by SDS-PAGE and immunoblotted (IB) with polyclonal anti-EGFR antibody (upper panel). The membrane was stripped and reprobed with an anti-c-Src monoclonal antibody (bottom panel). Comparable amounts of proteins were detected in immunoprecipitates of all the above experiments. The autoradiographs represent the results of a single experiment, which was carried out at least three times. Bar graphs represents intensities of associated EGFR quantified by scanning densitometry of blots and are expressed as a percentage relative to control with unstimulated as 100%. Values are represented as mean7s.e.m. of three independent experiments. Statistical comparisons were made with a one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test. Statistical significance: ***Po0.001, *Po0.5 versus unstimulated control cells.

of both angiotensin II- and arachidonic acid-induced ERK activation in renal proximal tubular epithelial cells. Several G protein-coupled receptors, including angiotensin II, have been presumed to induce activation of Src; however, the mechanisms have not been determined. In the present study, angiotensin II increased both c-Src phosphorylation and activity in a manner that is temporally consistent with a role of c-Src in mediating angiotensin II-induced ERK activity. In addition, there appears to be an angiotensin II AT2-receptor-mediated mechanism for activation of c-Src, wherein AT2-receptor blockade completely inhibited angiotensin II-induced c-Src activation, whereas AT1-receptor blockade did not. This is in discordance with a study in opossum kidney cells demonstrating that angiotensin II activated c-Src via activation of the AT1 receptor.19 Similar requirements for involvement of angiotensin II-stimulated c-Src activity have been demonstrated in smooth muscle cells,9,20–22 hepatocytes,23 cardiac fibroblasts,24 and cardiac myocytes.25 Together, these experiments indicate that the downstream actions of the AT2 receptor may be highly species dependent (i.e., opossum versus rabbit) and/or cell 1829

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Control

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Figure 13 | Arachidonic acid-, angiotensin II-, and EGF-induced increased association of EGFR with the c-Src SH2 domain, but not the c-Src SH3 domain. Serum-starved proximal tubular cells were treated in either the absence or presence of arachidonic acid (AA, 15 mM), angiotensin II (Ang II, 1 mM) or EGF (10 ng/ml) for 5 min at 371C. Cell lysates were immunoprecipitated (IP) with GST-Sepharose, GST-Src SH2- or GST-Src SH3-Sepharose as described in Materials and methods. Precipitated proteins (500 mg) were resolved by SDS-PAGE and immunoblotted (IB) with polyclonal anti-EGFR antibody (upper panel). The membrane was stripped and reprobed with an anti-c-Src monoclonal antibody (bottom panel). Comparable amounts of proteins were detected in immunoprecipitates of all the above experiments (data not shown). A representative autoradiogram from at least three independent experiments is shown. Bar graphs with error bars represent the mean7s.e.m. (n ¼ 3). ***Po0.001 indicates a significant increase in EGFR tyrosine phosphorylation due to its association with either c-Src SH2 or SH3 domain by treated cells versus the same matched agonist-stimulated GST cells.

type (i.e., vasculature versus epithelial and primary versus cell lines) specific. In addition, we present experimental evidence for an angiotensin II AT2-receptor-mediated mechanism for activation of c-Src, involving PLA2 activation and arachidonic acid release. Mepacrine and MAFP, two structurally unrelated cPLA2 inhibitors, significantly blocked angiotensin II-induced c-Src activation, suggesting that arachidonic acid is involved in angiotensin II-induced c-Src activation. Conversely, inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on angiotensin IIinduced c-Src activation, indicating that c-Src activation is dependent of cPLA2 enzyme activity in rabbit proximal tubule cells. Moreover, arachidonic acid itself induced timeand dose-dependent phosphorylation of c-Src, supporting the importance of PLA2 as a mediator of angiotensin II signaling. In addition, the effects of both angiotensin II and arachidonic acid on c-Src phosphorylation were independent of eicosanoid biosynthesis in that inhibitors of cyclooxygenase, lipoxygenase, and cytochrome P450 isoenzyme were without effect. Collectively, these observations document a mechanism of angiotensin II-induced c-Src activation, mediated by cPLA2-dependent release of arachidonic acid independent of production of epoxy derivatives of arachidonic acid. Angiotensin II-induced ERK activation in many other cell types requires an Src-dependent activation of the EGFR.26–29 1830

This supports the assumption that c-Src acts upstream of the EGFR. In the present study, we showed that stimulation of proximal tubule cells with angiotensin II resulted in significant EGFR tyrosine phosphorylation and its association with c-Src. Moreover, arachidonic acid mimicked these effects in a manner similar to angiotensin II. In addition, both angiotensin II- and arachidonic acid-induced EGFR tyrosine phosphorylation were uniformly inhibited by the Src family kinase inhibitor PP2 and by transient overexpression of a dominant-negative mutant of c-Src, thereby suggesting that the activation of c-Src might be important for the activation of ERK in proximal tubule cells exposed to angiotensin II and arachidonic acid. By comparison, neither PP2 nor dominant-negative c-Src effected EGF-induced EGFR tyrosine autophosphorylation or EGF-induced ERK activation. Interestingly, c-Src activation is not inhibited by the EGFR kinase inhibitor AG1478, suggesting that the EGFR is downstream of c-Src activation. Although arachidonic acid stimulated activation of other members of the Src family kinases, only c-Src was specifically co-immunoprecipitated with the EGFR. The activation of c-Src in proximal tubules by arachidonic acid is novel. This is in stark contrast to those results obtained in LLCPK1 cells, whereby arachidonic acid was ineffective in inducing EGFR tyrosine phosphorylation and c-Src activation. However, Chen et al.30,31 demonstrated that arachidonic acid induced significant increases in EGFR tyrosine phosphorylation and association with c-Src in F87V BM-3 transfected cells (regio- and stereoselective for 14(S), 15(R)-EET) that was inhibited by 17-octadecynoic acid, whereas no such effects were observed in vector-transfected cells. Thus, their experimental results suggested an eicosanoid-dependent tyrosine phosphorylation of EGFR and c-Src activation in LLCPK1 cells. This too is in discordance to our studies, whereby we consistently observed significant tyrosine phosphorylation of EGFR and increases in c-Src activity with exogenous arachidonic acid independent of eicosanoid biosynthesis. These differences between the two reports may be due to differences in either culture conditions or cell type specificity. Taken together, our findings provide strong support for the contention that Src family kinases play an important role in mediating EGFR transactivation by arachidonic acid and angiotensin II. They also suggest a specific role for c-Src in the arachidonic acid and angiotensin II stimulation of ERK activity, but they do not exclude contributions by other Src family kinases expressed in proximal tubule cells, particularly Fyn and Yes. Thus, further studies are required to explore the roles, if any, for these Src family kinases as mediators of arachidonic acid- and angiotensin II-induced MAP kinase activation. Both the kinase and the SH2 domain of Src are considered necessary for agonist-induced activation of receptor protein tyrosine kinases.32–35 Our findings show that both angiotensin II and arachidonic acid induced the association of the SH2 domain of c-Src with the EGFR in a dose- and timedependent manner that was significantly abrogated by PP2 or by transient overexpression of a dominant-negative c-Src Kidney International (2006) 69, 1823–1832

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LD Alexander et al.: Arachidonic acid induces c-Src activation

mutant. The effect of angiotensin II and arachidonic acid on EGFR tyrosine phosphorylation and its association with Src SH2 domain was similar to the effect of 10 ng/ml EGF. In contrast, the c-Src SH3 domain did not interact directly with tyrosine-phosphorylated EGFR. This is in agreement with a previous study demonstrating that lysophosphatidic acid, a G protein-coupled receptor agonist, induces the ability of the c-Src–SH2 domain GST fusion protein (but not the SH3 domain) to precipitate the tyrosine-phosphorylated EGFR.33 Moreover, the effects were mimicked by ETYA (a nonmetabolized stable analog of arachidonic acid which blocks arachidonic acid oxidation by acting as a false substrate),16,17 linoleic acid, and linolenic acid, suggesting that downstream eicosanoid metabolites do not mediate the effects. Thus, association of Src SH2 domain with the EGFR appears to be only triggered by polyunsaturated, long-carbon chain fatty acids (e.g., arachidonic acid, linoleic acid, and linolenic acid), whereas the association is not affected by short carbon chain (e.g., palmitoleic acid) or saturated (e.g., stearic acid and arachidic acid) fatty acids. The relevance of the present finding is the fact that arachidonic acid represents a novel mechanism whereby G protein-coupled receptors are linked to receptor and non-receptor protein tyrosine kinases in epithelial cells. Moreover, these studies are both timely and significant, wherefore they may have the potential to define a new mechanism for G protein-coupled receptor-induced c-Src/EGFR-mediated downstream signaling, that is through arachidonic acid, a common lipid second messenger.

Cell culture Primary culture of proximal tubule cells were isolated from male New Zealand white rabbits and maintained in standard growth media as described previously.14 Transient. transfection of dominant-negative c-Src The cells were transfected with 10 mg of plasmid DNA using the Lipofectamine 2000 reagent according to the manufacturer’s instructions. For mock transfection, the pCMV5 vector without the cDNA inserts was employed. c-Src kinase assay, immunoprecipitation, and Western blotting c-Src kinase activity was determined by using a Src kinase assay kit (Upstate Biotechnology) according to the manufacturer’s protocol. Briefly, 500 mg of protein was incubated with 1 mg of anti-c-src antibody 327 for 2 h at 41C, and the immune complexes were recovered with protein A Plus G Sepharose (Santa Cruz Laboratories Inc.). The activity was determined by phosphorylation of 10 ml Src kinase substrate peptide (No. 12–140) in Src kinase reaction buffer and 10 ml of [g32P]ATP. Immunoprecipitation and Western blotting were performed as described previously.14 Protein contents were determined by bicinchoninic acid assay. Statistical analysis The results are expressed as means7s.e.m. from combined experiments. The data were evaluated by one-way analysis of variance followed by Newman–Keuls multiple-comparison test or by paired Student’s t-test when appropriate. The limit of significance was a P-value p0.05. ACKNOWLEDGMENTS

MATERIALS AND METHODS Materials Arachidonic acid was obtained from ICN Pharmaceuticals (Costa Masa, CA, USA). (Sar1)-angiotensin II, bradykinin, PD123319, ETYA, and fatty acids (linoleic, linolenic, oleic, palmitoleic, stearic, and arachidic acids) were obtained from Sigma (St Louis, MO, USA). Human EGF, LipofectAMINE 2000, cell culture medium, and additives were obtained from Gibco BRL Life Technologies (Grand Island, NY, USA). PP2, AG1478, mepacrine, MAFP, haloenol lactone suicide substrate, horseradish peroxidase-conjugated goat antimouse, and rabbit immunoglobulin G were obtained from Calbiochem (La Jolla, CA, USA). [g32P]ATP was obtained from Dupont/NEN (Boston, MA, USA). c-Src (Src 2), Lck (2102), Lyn (44) Fyn (15) and c-Yes (3) rabbit polyclonal antibodies, protein A-, and protein A/G Plus-agarose beads were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-EGFR sheep polyclonal antibody, anti-phosphotyrosine (PY20) monoclonal antibody, and rabbit anti-sheep immunoglobulin G were obtained from Upstate Biotechnology (Lake Placid, NY, USA). Phospho-specific p44/42 MAP kinase (Thr202/Tyr204 polyclonal antibody) was purchased from Cell Signaling Technology (Beverly, MA, USA). Phosphospecific c-Src Tyr418 polyclonal antibody was purchased from Biosource International (Camarillo, CA, USA), and glutathione Sepharose 4B beads were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Losartan (DuP 753) was the generous gift of Dupont-Merck (White house station, NJ, USA). Candesartan was a kind gift of AstraZeneca (Mo¨lndal, Sweden). Dominant-negative c-Src K298M was a kind gift from Dr ChungHo Chang (Case Western Reserve University). Kidney International (2006) 69, 1823–1832

This work was supported by NIH-NHLBI grants HL 04363 (LD Alexander) and HL 41618 (JG Douglas). Dr Alexander is a National Center on Minority Health and Health Disparities (NCMHD) Scholar. REFERENCES 1.

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