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Endocrinology Vol. 142, No. 2 564-572
Copyright © 2001 by The Endocrine Society

ARTICLES

Adrenomedullin Stimulates Proline-Rich Tyrosine Kinase 2 in Vascular Smooth Muscle Cells

Hiroaki Iwasaki, Masayoshi Shichiri, Fumiaki Marumo and Yukio Hirata

Department of Clinical and Molecular Endocrinology, Graduate School, Tokyo Medical and Dental University, Tokyo 113-0034, Japan

Address all correspondence and requests for reprints to: Yukio Hirata, M.D., Ph.D., Department of Clinical and Molecular Endocrinology (the Second Department of Internal Medicine), Graduate School, Tokyo Medical and Dental University, 1–5-45, Yushima, Bunkyo-ku, Tokyo 113-0034, Japan. E-mail: yhirata.cme{at}tmd.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A novel vasodilator peptide, adrenomedullin (AM) stimulates extracellular signal-regulated kinase (ERK) 1/2 via yet uncharacterized 120 kDa tyrosine kinase(s) in rat vascular smooth muscle cells (VSMC). In the present study, we have examined whether the AM-activated tyrosine kinase is proline-rich tyrosine kinase 2 (PYK2) associable with adapter proteins. AM rapidly (within 30 sec) and dose dependently increased tyrosine kinase activity, whose effect was enhanced in the presence of o-vanadate, a phosphatase inhibitor. A tyrosine kinase with an apparent molecular mass of 120 kDa corresponding to that of PYK2 was predominantly localized to the cytosolic fraction, whereas the tyrosine-phosphorylated 180-kDa protein was observed in the membrane fraction from EGF-treated cells, but not from AM-treated cells. AM induced rapid (within 30 sec) and transient phosphorylation of PYK2, but not focal adhesion kinase. AM caused autophosphorylation of tyrosine residue(s) of PYK2 and promoted its association with adaptor proteins (Shc/Grb2). AM rapidly (within 1 min) activated c-Src and enhanced its association with tyrosine-phosphorylated PYK2. These data suggest that AM stimulates PYK2 which, in turn, activates c-Src and induces recruitment of adaptor proteins (Shc/Grb2), thereby leading to activation of p21ras/ERK1/2 cascade in VSMC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADRENOMEDULLIN (AM) is a novel vasorelaxant peptide with a partial structural homology to the calcitonin-gene related peptide (CGRP) (1). Three putative receptors with seven-transmembrane domains that bind to AM and/or CGRP have been reported: L1 (2), RDC1 (3) and calcitonin receptor-like receptor (CRLR) (4). Recently, AM is shown to be produced by and secreted from vascular smooth muscle cells (VSMC), endothelial cells (EC) and activated macrophages in atherosclerotic lesions (5), suggesting its possible involvement not only in the regulation of vascular tone, but also in the process of atherosclerosis. However, it remains elusive whether these receptors and/or other unidentified receptors act on VSMC.

One of the major signal pathways used by AM is considered to be the adenylate cyclase/cAMP system for its vasodilatory action (6). In several cell types other than VSMC, however, AM activates other signaling pathways, such as phospholipase C (7) and guanylate cyclase (7, 8). Furthermore, we have recently demonstrated that AM stimulated extracellular signal-regulated kinase (ERK) 1/2 activity independently from adenylate cyclase/cyclic AMP system in rat VSMC (9). AM has also been shown to activate other mitogen-activated protein (MAP) kinase family, such as p38 MAP kinase and c-Jun NH2-terminal protein kinase (JNK) in rat mesangial cells (10). However, the initial signaling event(s) leading to the activation of MAP kinase family in response to AM still remains unknown.

Proline-rich tyrosine kinase 2 (PYK2), also known as cell adhesion kinase (CAK) ß, related adhesion focal tyrosine kinase (RAFTK) or calcium-dependent tyrosine kinase (CADTK), is a focal adhesion kinase (FAK)-related cytoplasmic tyrosine kinase (11, 12). PYK2 has been shown to be abundantly expressed in various tissues including brain, lung, intestine, kidney, spleen (13, 14), and VSMC (15, 16, 17). PYK2 is activated by various extracellular stimuli, such as G protein-coupled receptor (GPCR) agonists, protein kinase C (PKC) activation, an increase in intracellular Ca2+ concentration ([Ca2+]i), UV irradiation and extracellular osmolarity (11, 12). Recently, we have demonstrated that angiotensin II (AII) stimulated PYK2 via increase in [Ca2+]i in rat VSMC (18).

PYK2, when autophosphorylated on tyrosine residues, provides binding sites for other Src homology (SH) 2 domain-containing proteins including Src tyrosine kinase family (19, 20). The association of PYK2 with c-Src leads to translocation of adapter proteins (Shc/Grb2) to the plasma membrane and subsequent p21ras-dependent ERK1/2 activation (20, 21). We have recently shown that AM stimulated tyrosine phosphorylation of several proteins among which tyrosine kinase(s) with an apparent molecular mass of 120 kDa (p120) can recruit adapter proteins (Shc/Grb2), thereby leading to p21ras-dependent ERK1/2 activation (9).

To elucidate the involvement of PYK2 in AM-induced ERK1/2 activation in rat VSMC, we investigated whether PYK2 actually contributes to the AM-induced tyrosine kinase cascade and causes the recruitment of adaptor proteins (Shc/Grb2) in rat VSMC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic rat AM was purchased from Peptide Institute (Osaka, Japan), epidermal growth factor (EGF) and AG1478 from Calbiochem-Nobabiochem (La Jolla, CA), agarose-conjugated glutathione-S-transferase (GST)-Grb2(1–217) fusion protein, polyclonal anti-PYK2, polyclonal anti-Src, polyclonal anti-Shc, antigoat horseradish peroxidase (HRP)-conjugated second antibodies and protein A/G-agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), monoclonal anti-phosphotyrosine (pTyr) (PY20) and monoclonal anti-PYK2 antibodies from Transduction Laboratories, Inc. (Lexington, KY), monoclonal anti-PYK2, monoclonal anti-FAK, monoclonal anti-pTyr (4G10) and monoclonal anti-Src (GD11) antibodies from Upstate Biotechnology, Inc. (Lake Placid, NY), antimouse and antirabbit HRP-conjugated second antibodies from Amersham Pharmacia Biotech (Buckinghamshire, UK), o-vanadate, poly(Glu) and poly(Glu80Tyr (20)) were from Sigma (St. Louis, MO). [{gamma}-32P]ATP (specific activity 37 MBq/mmol) were also purchased from Amersham Pharmacia Biotech.

Cell culture
VSMC were prepared from the thoracic aorta of 12-week-old-male Sprague Dawley rats by the explant method and cultured in DMEM containing 10% FCS at 37 C in a humidified atmosphere of 95% air-5% CO2 as described previously (9). Quiescent VSMC (5–15th passages) after 48–72 h serum-starvation were used in the following experiments.

Preparation of subcellular fraction
Quiescent VSMC preincubated with 200 µMo-vanadate for 15 min were stimulated with either AM or EGF for 1 min. Cells were scraped with a disposable cell-scraper in ice-cold lysis buffer [20 mM HEPES (pH 7.3) containing 500 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM Na3VO4, 10% glycerol, 10 µg/ml phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, and 100 KIU/ml aprotinin], and homogenized with a Teflon-glass homogenizer (Kinematica GmbH Littau, Switzerland) at 4 C. The homogenates were ultracentrifuged at 105,000 x g for 45 min at 4 C. The membrane fraction was resuspended in the same buffer with Triton X-100; Trtion X-100 was also added to each portion of the original homogenate and the cytosolic fraction. Each sample was subjected to immunoblotting using anti-pTyr antibody.

Immunoprecipitation and immunoblotting
Cells were lysed with ice-cold lysis buffer [20 mM HEPES (pH 7.3) containing 500 mM NaCl, 50 mM NaF, 5 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 10 µg/ml PMSF, 20 µg/ml leupeptin, and 100 KIU/ml aprotinin], and the lysates were clarified by centrifugation at 14,000 x g for 5 min at 4 C. The supernatants were immunoprecipitated with either 4 µg each of anti-pTyr (PY20), anti-PYK2, anti-Shc or anti-Src antibody, followed by the addition of 30 µl each of protein A/G agarose or GST-Grb2 fusion protein. After incubation for 4 h at 4 C, immune complexes were collected by centrifugation at 14,000 x g for 1 min at 4 C, washed with either lysis buffer for immunoblotting or 2 fold-concentrated tyrosine kinase assay buffer [100 mM HEPES (pH 7.6) containing 60 mM MgCl2, 2 mM MnCl2, 0.2 mM Na3VO4, 0.2% Nonidiet P-40] for immune complex kinase and autophosphorylation assay.

For immunoblotting, aliquots of the washed immune complex suspensions were solubilized in 1x sample buffer [62.5 mM Tris-HCl (pH6.8) containing 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue] or 5x sample buffer, boiled for 5 min, and then subjected to SDS-PAGE. Proteins in the gel were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech) by electroblotting. The membrane was treated with either anti-pTyr, anti-PYK2, anti-FAK, anti-Shc or anti-c-Src antibody, followed by incubation with HRP-conjugated second antibody; immunoreactive proteins were detected by an ECL system as described (9).

Immune complex kinase assay
Aliquots of the washed immune complex suspensions were preincubated for 5 min at 4 C with either 160 µg of synthetic tyrosine kinase substrate poly[Glu80Tyr (20)]or control substrate poly(Glu) for tyrosine kinase and PYK2 activities, and with a synthetic p34(6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) peptide (KVEKIGEGTYGVVYK) as a substrate for Src activity, respectively. After incubation with 5 µCi [{gamma}-32P]ATP for 4 min at 25 C for tyrosine kinase and PYK2 activity or with 10 µCi [{gamma}-32P]ATP for 10 min at 30 C for c-Src activity, samples were spotted onto P81 phosphocellulose paper (Whatman, Maidstone, UK). The papers were washed twice with trichloroacetic acid (TCA), air-dried, and assayed by a liquid scintillation counter for acid-insoluble [32P].

Autophosphorylation and phosphoamino acid analysis
Aliquots of immune complex suspensions of PYK2 were incubated with 25 µCi [{gamma}-32P] ATP for 10 min at 25 C, followed by addition of 5x sample buffer, and boiling for 5 min, and then subjected to SDS-PAGE. Proteins in the gel were transferred to a Immobilon-P membrane (Millipore Corp., Bedford, MA) by electroblotting. Radioactive molecules were determined with an imaging analyzer (BAStation system, Fuji Photo Film Co., Ltd., Tokyo, Japan). The membranes containing the [32P]-labeled species of interest were excised and subjected to acid hydrolysis followed by two dimensional double chromatography as described (22, 23); [32P]-labeled phosphoserine, phosphothreonine and pTyr were localized by comigration with ninhydrin-stained standards.

DNA synthesis
DNA synthesis was assessed by incorporation of [3H]thymidine into cells as reported (9). In brief, quiescent VSMC pretreated with or without AG1478 (250 nM) for 30 min were incubated with AM (100 nM) at 37 C for 20 h in serum-free DMEM, after which 1 µCi [3H]thymidine was added, and the cells were further incubated for 4 h. After completion, TCA-insoluble radioactivity was measured in a liquid scintillation counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AM activates tyrosine kinases and tyrosine-phosphorylates 120 kDa protein(s)
We previously demonstrated that AM rapidly induced tyrosine phosphorylation of several proteins, among which tyrosine-phosphorylated p120 recruited adaptor proteins (Shc/Grb2) (9). To ascertain that AM activates tyrosine kinases in rat VSMC, the catalytic activity of tyrosine kinases was assessed by immune complex tyrosine kinase assay (Fig. 1Go). AM (100 nM) caused a transient activation of tyrosine kinases, peaking at 1 min, followed by a decline to the basal level by 10 min (Fig. 1AGo), whose effect was dose-dependent (1 nM-1 µM) with an approximate EC50 of 2.2 nM (Fig. 1BGo).



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  		Figure 1. Time- and dose-dependent activation of tyrosine kinases by AM in rat VSMC. Cells were stimulated (A) with AM (100 nM) for the indicated times, and (B) in the indicated concentrations for 1 min. Cell lysates were immunoprecipitated with anti-phosphotyrosine (pTyr) antibody and subjected to immune complex kinase assay. Tyrosine kinase activity of the anti-pTyr immune complex was determined; each point represents the mean ± SEM (n = 3).

 
To determine the involvement of tyrosine-phosphorylated p120 in AM-activated tyrosine kinases, the pTyr content of p120 after AM treatment was assessed by immunoprecipitation with anti-pTyr antibody followed by immunoblotting with anti-pTyr antibody (Fig. 2Go). Similar to the time-course of AM-induced activation of tyrosine kinases, AM rapidly (within 30 sec) and transiently phosphorylated p120 (Fig. 2AGo), whose effect was also dose-dependent (1 nM-1 µM) (Fig. 2BGo).



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  		Figure 2. Tyrosine phosphorylation of 120 kDa protein by AM. Cells were stimulated (A) with AM (100 nM) for the indicated times, (B) in the indicated concentrations for 1 min, and (C) after pretreatment with or without o-vanadate (200 µM) for 15 min, stimulated with AM (100 nM) for 1 min. Cell lysates were immunoprecipitated with anti-pTyr antibody, and then immunoblotted with anti-pTyr antibody. An arrowhead indicates tyrosine-phosphorylated protein(s) with molecular size of 120 kDa.

 
AM-stimulated tyrosine phosphorylation occurs independent of tyrosine phosphatase activity
The AM-stimulated tyrosine phosphorylation may be due to inhibition of protein tyrosine phosphatase(s), because another GPCR agonist, endothelin-1 (ET-1) has been shown to stimulate tyrosine phosphorylation of FAK by suppressing protein tyrosine phosphatase activity (24). To determine the possible involvement of tyrosine phosphatase in AM-stimulated tyrosine phosphorylation, the effect of a phosphatase inhibitor, o-vanadate was examined. O-vanadate (200 µM), when added alone, increased tyrosine phosphorylation of p120, and further enhanced AM-stimulated tyrosine phosphorylation (Fig. 2CGo). Therefore, the AM-stimulated tyrosine phosphorylation occurs independent of tyrosine phosphatase activity.

AM-induced tyrosine-phosphorylated 120 kDa protein(s) is localized in the cytosol
To characterize the tyrosine-phosphorylated 120-kDa tyrosine kinase(s), we next examined the subcellular localization of these tyrosine-phosphorylated proteins by ultracentrifugation followed by immunoblotting with anti-pTyr antibody (Fig. 3AGo). Several tyrosine-phosphorylated proteins (60, 80, 90, 120 kDa) after AM treatment were exclusively localized to the cytosolic fraction in the same fashion as those after EGF treatment. In contrast, the tyrosine-phosphorylated 180-kDa protein was observed in the membrane fraction from EGF-treated cells, but not from AM-treated cells, which may represent autophosphorylated EGF receptor.



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  		Figure 3. A, Subcellular fraction of phosphorylated tyrosine substrates by AM. Cells were stimulated with or without either EGF (100 ng/ml) or AM (100 nM) for 1 min. After ultracentrifugation of cell homogenates, each subcellular fraction (total homogenate, cytosol, or membrane) was subjected to immunoblotting using anti-pTyr antibody. An arrowhead and an arrow indicate 120 kDa and 180 kDa tyrosine-phosphorylated protein(s), respectively. B, Effect of AG1478 on AM-induced DNA synthesis. After treatment with (•) or without ({circ}) AG1478 (250 nM) for 30 min, cells were incubated with or without AM (100 nM) for 20 h; [3H]thymidine incorporated during 4 h were measured. Each column with bar represents the mean ± SEM (n = 3).

 
It has recently been reported that several GPCR agonists, such as AII and ET-1, stimulate tyrosine phosphorylation of EGF receptor via ligand-independent transactivation mechanism in rat VSMC, thereby leading to ERK1/2 activation and mitogenesis (25, 26). To determine whether transactivation of EGF receptor participates in growth-promoting effect by AM, the effect of an EGF receptor kinase inhibitor, AG1478 (27), on AM-stimulated DNA synthesis was examined (Fig. 3BGo). AG1478 (250 nM) only partially inhibited the AM-stimulated incorporation of [3H]thymidine, suggesting that EGF receptor transactivation is not involved in AM-stimulated VSMC growth.

AM causes tyrosine phosphorylation and activation of PYK2
Because the molecular weight of the AM-induced tyrosine kinase(s) was approximately 120 kDa and localized exclusively in the cytosolic fraction, we assumed that the tyrosine kinase(s) belongs to the nonreceptor cytosolic tyrosine kinase family. Therefore, we next examined whether AM activates PYK2 and/or FAK by immunoprecipitation with either anti-PYK2 or anti-FAK antibody followed by immunoblotting with anti-pTyr antibody (Fig. 4Go). AM rapidly (within 30 sec) tyrosine-phosphorylated PYK2 (Fig. 4AGo), but not FAK (Fig. 4BGo). AM transiently (0.5–2 min) activated PYK2 which gradually declined to basal levels by 5 min as determined by immune complex kinase assay (Fig. 4CGo). These data strongly suggest that the AM-activated p120 tyrosine kinase is PYK2, but not FAK.



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  		Figure 4. Time-courses of tyrosine kinase activities of PYK2 and FAK by AM. Cells were stimulated with AM (100 nM) for the indicated times. Cell lysates were immunoprecipitated with either (A) anti-PYK2 or (B) anti-FAK antibody, and then immunoblotted with either anti-pTyr, anti-PYK2 or anti-FAK antibody, respectively. Arrowheads indicate tyrosine-phosphorylated PYK2 and FAK. C, Time-dependent PYK2 activation by AM. Tyrosine kinase activity of anti-PYK2 immune complex was determined; each point represents the mean ± SEM (n = 3).

 
AM autophosphorylates PYK2
Receptor and nonreceptor tyrosine kinases, when activated, are autophosphorylated, thereby providing binding sites for downstream target molecules containing SH2 domain, such as c-Src and Grb2. To examine whether the phosphorylation of PYK2 is responsible for autophosphorylation and/or activation of an intermediating kinase, autophosphorylation assay was performed (Fig. 5Go). AM rapidly (within 1 min) enhanced autophosphorylation of PYK2 (Fig. 5AGo) with a time-course similar to that of tyrosine kinase activity (data not shown). Furthermore, phosphoamino acid analysis revealed the presence of phosphorylated tyrosine residues, but not serine or threonine residues of phosphorylated PYK2 (Fig. 5BGo). These data indicate that AM-phosphorylated and activated PYK2 with an intrinsic and autophosphorylating tyrosine kinase activity.



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  		Figure 5. Autophosphorylation of PYK2 by AM. A, Cell lysates treated with or without AM (100 nM) for 1 min were immunoprecipitated with anti-PYK2 antibody. Aliquots of the immune complex suspension were incubated in the presence of [{gamma}-32P] ATP, subjected to SDS-PAGE, and transferred to Immobilon-P. An arrowhead indicates autophosphorylating PYK2. B, The regions of Immobilion-P containing the [32P]-labeled species from anti-PYK2 immunoprecipitates were excised and subjected to phosphoamino acid analysis. Locations of ninhydrin-stained phosphoserine (PS), phosphothreonin (PT), and pTyr (PY) standards are indicated.

 
AM stimulates association of tyrosine-phosphorylated PYK2 with adaptor proteins
The association of adapter proteins (Shc/Grb2) with tyrosine-phosphorylated protein plays a critical role to recruit Sos, a GDP/GTP exchange factor of p21ras. Therefore, we examined whether AM induces the association of PYK2 with these adapter molecules using immunoprecipitation with anti-Shc antibody and coprecipitation with GST-Grb2 fusion protein followed by immunoblotting with either anti-pTyr, anti-PYK2 or anti-Shc antibody (Fig. 6Go). AM rapidly (within 1 min) increased association of the tyrosine-phosphorylated PYK2 with Shc (Fig. 6AGo) and Grb2 (Fig. 6BGo); three tyrosine-phosphorylated Shc isoforms (p66, p52, p46) were concomitantly associated with Grb2 after stimulation with AM (Fig. 6BGo, lower panel). These data suggest that tyrosine-phosphorylated PYK2 by AM is mediated via its signaling complex with adapter proteins (Shc/Grb2).



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  		Figure 6. Association of tyrosine-phosphorylated PYK2 and adapter proteins by AM. Cells were stimulated with or without AM (100 nM) for 1 min. Cell lysates were precipitated with (A) anti-Shc antibody and (B) GST-Grb2 fusion protein, and then immunoblotted with either anti-pTyr, anti-PYK2 or anti-Shc antibody, respectively. Arrowheads denote tyrosine-phosphorylated PYK2 (upper and middle panel) and three Shc isoforms (lower panel), respectively.

 
AM stimulates association of c-Src with PYK2
A major autophosphorylation site (Tyr402 of PYK2 is recognized by the SH2 domain of c-Src tyrosine family, and the PYK2-induced activation of c-Src is necessary for phosphorylation of Shc and its association with Grb2 as well as for the subsequent activation of ERK1/2 cascades (20). To determine whether AM can activate c-Src to form signaling complex with PYK2, we performed c-Src kinase assay and immunoprecipitation with anti-PYK2 antibody followed by immunoblotting with either anti-pTyr, anti-PYK2 or anti-c-Src antibody (Fig. 7Go). AM caused a transient activation of c-Src, peaking at 2 min, which returned to the basal level by 10 min (Fig. 7AGo). Furthermore, AM rapidly (within 1 min) stimulated the association of c-Src with PYK2, with a time-course similar to that of c-Src activation (Fig. 7BGo). Therefore, it is likely that AM-activated c-Src is responsible for the formation of signaling complex between PYK2 and adaptor molecules (Shc/Grb2).



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  		Figure 7. Time-dependent association of c-Src with PYK2 by AM. A, Cells were stimulated with AM (100 nM) for the indicated times. Cell lysates were precipitated with anti-c-Src antibody and subjected to immune complex Src assay; each point represents the mean ± SEM (n = 3). (B) Cell lysates were immnoprecipitated with anti-PYK2 antibody, and then immunoblotted with either anti-pTyr, anti-PYK2 or anti-c-Src antibody, respectively. An arrowhead denotes tyrosine-phosphorylated PYK2 (upper and lower panel) and c-Src (middle panel), respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study clearly demonstrated that AM markedly induced activation of tyrosine kinases in rat VSMC. Although the exact AM receptors in VSMC are not characterized yet, the predicted amino acid structures of the putative AM/CGRP receptors, L1 (2), RDC1 (3, 28), and CRLR (29, 30), have no tyrosine kinase domains. However, the rapid and dose-dependent tyrosine phosphorylation by AM in the present study suggests that AM directly activates tyrosine kinase and/or inhibits tyrosine phosphatase activity. Our result that the effects on tyrosine phosphorylation by AM and o-vanadate, a tyrosine phosphatase inhibitor, were additive strongly suggests that AM-induced tyrosine phosphorylation is due to its direct activation of tyrosine kinase rather than inhibition of phosphatase activity.

Recent studies revealed that a variety of stimuli, such as GPCR agonists, cytokines, irradiation, and osmotic stress, induce ERK1/2 activation via phosphorylation and activation of receptor tyrosine kinases in various cell types including VSMC (31). We have recently demonstrated that EGF receptor transactivation by AII occurred downstream to PYK2 in VSMC (18). The present subfraction study revealed that a set of tyrosine-phosphorylated proteins with different molecular sizes (~60, ~80, ~90, and ~120 kDa) in the cytosolic fraction after stimulation with AM and EGF were common. However, the tyrosine-phosphorylated p180 abundant in the membrane fraction after EGF stimulation, presumably phosphorylated EGF receptor, was not detected after AM stimulation. These data are compatible with our previous data showing that phosphorylated ~180-kDa protein was not coprecipitatable with GST-Grb2 fusion protein from AM-treated VSMC (9). Furthermore, AG1478, a selective EGF receptor kinase inhibitor, which completely blocked the hypertrophic effects by AII (25) and ET-1 (26), had only limited effect on AM-stimulated DNA synthesis. Therefore, it is suggested that transactivation of EGF receptor is not involved in signal transduction by AM in VSMC.

The present results showed that AM-stimulated phosphorylated 120-kDa protein was predominantly localized to the cytosolic fraction, suggesting it as a member of cytosolic and nonreceptor tyrosine kinase family. Indeed, our present study revealed that AM caused a time-dependent tyrosine phosphorylation of PYK2, a 120-kDa cytosolic and nonreceptor tyrosine kinase. A number of receptor and nonreceptor tyrosine kinases including PYK2, when activated, have been shown to be autophosphorylated (11, 12). Our present study clearly revealed that AM enhanced autophosphorylation of PYK2 and increased its phosphorylated tyrosine content. Thus, our study provide a direct evidence that AM stimulates PYK2 by activating its intrinsic and autophosphorylating kinase.

It should be noted that approximately 120 kDa tyrosine-phosphorylated proteins are not only restricted to PYK2, but also FAK as a potential candidate, although this possibility was excluded by the immunoprecipitation experiment. It has been reported that PYK2 and FAK are differentially regulated in VSMC. For example, tyrosine phosphorylation of PYK2 was more rapidly and influenced by soluble factors, whereas that of FAK was slow and primarily controlled by cell adhesion (15, 17). However, it has been recently shown that PYK2 directly cross-phosphorylated FAK, which acted as a target for the SH2 domain containing proteins (32). Therefore, it remains to be determined whether AM indirectly affects signal transduction of FAK through PYK2.

It is well established that translocation of adapter proteins/Sos to p21ras localized on plasma membrane is a key event in regulation of the ERK1/2 pathway. In the case of PYK2, translocation of the Grb2/Sos complex is mediated via direct binding of the SH2 domain of Grb2 to Tyr881 of PYK2 and/or indirect association with c-Src and Shc (20). The present study revealed that AM not only induced the association of PYK2 with Shc and Grb2, but also activated c-Src tyrosine kinase, thereby leading to formation of PYK2/c-Src complex. The AM-stimulated activation of c-Src was transient with a maximal tyrosine phosphorylation at 2 min, suggesting that the early phase of c-Src activation may be sufficient to induce the binding of Shc/Grb2 complex to PYK2. Taken together, tyrosine-phosphorylated PYK2 by AM mainly recruits Grb2 after tyrosine phosphorylation of c-Src and Shc as illustrated in Fig. 8Go. Because a Grb2 mutant with a deletion of the amino-terminal tail of SH3 domain reduced PYK2-induced ERK1/2 activation (20), it is conceivable that the AM-induced PYK2 activation may lead to p21ras-dependent ERK1/2 activation through activation of c-Src and recruitment of adaptor proteins in VSMC. An alternative signaling pathway of PYK2 may involve other downstream transducers. For example, it has been shown that Grb2/Sos complex connected PYK2 to the activation of ERK1/2, whereas other adapter proteins, such as p130Cas and Crk, linked PYK2 with JNK pathway (20). Further studies are required to determine the interaction of PYK2 with other signaling molecules by AM. It also remains to be determined how the activation of AM receptors leads to PYK2 activation and phosphorylation.



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  		Figure 8. Possible molecular mechanism of VSMC growth stimulated by AM. AM induces PYK2 activation, resulting in activation of c-Src and recruitment of adaptor proteins (Shc, Grb2 and Sos). Then, Sos catalyzes the exchange of GDP to GTP on p21ras, leading to activation of ERK1/2 cascade and ultimately growth promotion of VSMC.

 
Although the exact role of tyrosine phosphorylation of PYK2 in AM signaling remains to be determined, a reversible phosphorylation of tyrosine kinases is known to be a regulatory machinery in cell growth and mitosis. In this regard, AM has recently been shown to be involved in regulation of development (33) and carcinogenesis (34, 35). In fact, both AM and CGRP stimulate DNA synthesis in EC (36), fibroblasts (37), and VSMC (9). Our previous study revealed that AM-induced ERK1/2 activation and cell proliferation were completely blocked by inhibitors of protein tyrosine kinases (9). Based on the observation that AM is secreted from EC, VSMC, and activated macrophages in atherosclerotic lesions (5), the AM-stimulated activation of PYK2 in VSMC as demonstrated in this study may play a pivotal role in the process of vascular remodeling in atherosclerosis.

In conclusion, we have demonstrated that AM induced PYK2 activation which causes activation of c-Src and recruitment of adapter proteins (Shc/Grb2), thereby leading to p21ras-dependent ERK1/2 activation in rat VSMC.


   Acknowledgments
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sport and Culture, Japan, and fund from Takeda Science Foundation.

Received June 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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