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(Circulation Research. 1997;81:550-557.)
© 1997 American Heart Association, Inc.

Articles

Angiotensin II Stimulates Tyrosine Phosphorylation of Phospholipase C-–Associated Proteins

Characterization of a c-Src–Dependent 97-kD Protein in Vascular Smooth Muscle Cells

Udo Schmitz, Mari Ishida, , Bradford C. Berk

From the Department of Medicine, Division of Cardiology, University of Washington, Seattle.

Correspondence to Dr Bradford C. Berk, University of Washington, Division of Cardiology, Box 357710, Seattle, WA 98195-7710. E-mail bcberk{at}u.washington.edu

	Abstract

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Abstract Stimulation of phospholipase C- (PLC-) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC- via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-–associated proteins, RASM cells were labeled with [³²P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC- was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-–associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src–transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-–associated protein that may be required for Ang II–mediated signal transduction.

Key Words: phospholipase C • angiotensin II • c-Src • smooth muscle • tyrosine kinase

	Introduction

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In VSMCs, the cellular effects of Ang II are mediated through the G protein–coupled AT₁ receptor.¹ The putative membrane structure of the AT₁ receptor indicates that it is a member of the seven transmembrane–spanning domain receptors that lack intrinsic tyrosine kinase activity.^{2 3} Nonetheless, binding of Ang II to the AT₁ receptor elicits many of the same cellular events induced by mitogens such as PDGF and EGF. The similarity in signal transduction induced by the AT₁ receptor and the PDGF and EGF receptors is most apparent in the stimulation of tyrosine phosphorylation of shared signaling molecules.^{4 5} Recently, we demonstrated that Ang II stimulation of PIP₂ hydrolysis in cultured RASM cells is mediated by PLC-. The time course for PLC--mediated PIP₂ hydrolysis was coincident with its tyrosine phosphorylation. A critical role for PLC- tyrosine phosphorylation was suggested by two experiments. First, inhibition of tyrosine phosphorylation with genistein, a tyrosine kinase inhibitor, prevented PIP₂ hydrolysis.⁶ Second, electroporation of Src antibodies into RASM cells led to the inhibition of Ang II–stimulated PLC- activation.⁷ We have recently shown that Ang II stimulates c-Src activity and that the time course for c-Src activation precedes PLC- activation, suggesting a regulatory role for c-Src.^{7 8} Although Src-kinase family members have been demonstrated to phosphorylate PLC-1 and PLC-2 in vitro, an association in vivo between c-Src and PLC- has not been observed in RASM cells (authors' unpublished data, 1996) or other cells.^{9 10} We hypothesized that the failure to demonstrate a direct interaction between PLC- and c-Src in vivo might be due to the existence of a linker protein that mediates their interaction.

Among several proteins suggested to mediate interactions between PLC- and regulatory proteins, we identified a tyrosine-phosphorylated 62-kD protein from the literature as a likely candidate. This p62 protein was shown to associate with PLC- by binding to its SH2 domains in murine C3H10T/2 fibroblasts.¹¹ Furthermore, this association was most readily demonstrated in cells that were transformed with v-Src or that overexpressed the human EGF receptor.¹¹ Phosphopeptide mapping showed that the PLC-–associated p62 protein was highly related to, if not identical to, a GAP-associated p62 protein that was initially described by Ellis et al.¹² GAP-associated p62 was shown to bind to Src-SH2 domains and GAP N-terminal SH2 domains in vitro and to be heavily phosphorylated in v-Src–transformed cells and cells overexpressing the EGF receptor.^{12 13} The GAP-associated p62 may also be identical to a 60-kD insulin receptor substrate identified by Hosomi et al¹⁴ and Ogawa and colleagues.^{15 16} These investigators generated a monoclonal antibody against a 60-kD protein that associated with Ras-GAP via binding to the N-terminal SH2 domain of Ras-GAP. This antibody (2C4) recognizes a nondenatured epitope only and thus cannot be used for Western blotting. Therefore, the true identities of GAP-associated p62, PLC-–associated p62, and insulin receptor substrate p60 cannot be proven, although it is generally assumed that they constitute a highly related set of proteins, if they are not identical. In the present study, we refer to this protein(s) as p60. To date, p60 has not been cloned, and its function remains unknown.

In the present study, we immunoprecipitated PLC- from RASM cells and characterized PLC-–associated proteins that were phosphorylated in response to Ang II. Using the 2C4 antibody, we found that p60 does not participate in Ang II–dependent activation of PLC- in RASM cells. In addition, we identified a PLC-–associated protein of 97 kD (termed p97) that was tyrosine-phosphorylated upon Ang II stimulation of RASM cells. Ang II–stimulated tyrosine phosphorylation of p97 appeared to be dependent on c-Src activity, as shown by decreased p97 tyrosine phosphorylation in VSMCs isolated from Src-/- mice. These findings suggest that p97 participates in Ang II–stimulated signal transduction involving c-Src and PLC-.

	Materials and Methods

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Materials
Monoclonal antibodies to PLC-1, anti-phosphotyrosine (4G10), and polyclonal anti-human PDGF type-B receptor antibody were purchased from Upstate Biotechnology. Monoclonal p120 Ras-GAP antibody (B4F8), anti-phosphotyrosine antibody PY20, and HRP-coupled PY20 were purchased from Santa Cruz Biotechnology Inc. Monoclonal Nck antibody was purchased from Transduction Laboratories. Monoclonal p60^src antibody (clone 327) was purchased form Oncogene Science. Monoclonal p60 antibody (2C4) was a kind gift of Dr R. Roth (Stanford University).¹⁵ Protein G agarose was obtained from GIBCO-BRL. HRP-coupled goat anti-mouse antibody, an ECL detection kit, and [³²P]orthophosphate were purchased from Amersham. CP-118,556 (PP1) was kindly provided by Pfizer Inc, Groton, Conn.¹⁷

Cell Culture
Aortic smooth muscle cells were isolated from 200- to 250-g male Sprague-Dawley rats, from Src-/- mice, and from the corresponding wild-type mice and maintained in DMEM supplemented with 10% bovine calf serum, as previously described. The Src-/- mice and corresponding wild-type mice were a kind gift of Sheila Thomas (Fred Hutchinson Cancer Research Center, Seattle, Wash).¹⁸ Passage-8 to -15 VSMCs at 80% confluence were growth-arrested by incubation in 0.4% calf serum for 48 hours before use. NIH-3T3 cells and v-Src–transformed NIH-3T3 cells were kindly provided by David Shalloway (Cornell University).¹⁹ They were grown under the same conditions as VSMCs, except that they were not growth-arrested before use.

Immunoprecipitation and Western Blot Analysis
Growth-arrested VSMCs or NIH-3T3 cells were either left untreated or were treated with 100 nmol/L Ang II for the indicated times. VSMCs were pretreated with the different tyrosine kinase inhibitors, as indicated in the figure legends. Cells were lysed with lysis buffer containing 20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, 20 mmol/L ß-glycerophosphate, 1 mmol/L sodium orthovanadate, 10 µg/mL leupeptin, and 1 mmol/L PMSF. Lysates were precleared by centrifugation, and protein concentration was measured by DC protein assay (Bio-Rad). The indicated antibodies were added to equal amounts of protein per sample and incubated for 12 hours at 4°C. Antibody complexes were collected by addition of protein G–agarose for 3 hours. Precipitates were washed five times in cell lysis buffer, resuspended in SDS sample buffer, and boiled for 10 minutes. After centrifugation for 10 minutes at 10 000g, the supernatants were size-fractionated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the indicated antibodies. Secondary antibodies were coupled to HRP, and Western blot detection was performed by ECL (Amersham). Equal loading of the immunoprecipitated protein of interest was ascertained in every experiment by Western blotting.

[³²P]Orthophosphate Labeling of VSMCs
RASM cells were labeled with [³²P]orthophosphate for 4 hours as previously described.⁶ RASM cells were then either left untreated or stimulated with 100 nmol/L Ang II for 5 minutes, and immunoprecipitation of PLC- was performed as described above. Protein samples were size-fractionated in large (14x14-cm) 7.5% SDS-polyacrylamide gels. The gels were either dried, or proteins were transferred to nitrocellulose membranes for 5 hours at a constant voltage of 50 V in a transfer system for large gels (Trans-Blot Cell, Bio-Rad). Phosphorylated proteins were visualized by autoradiography. Nitrocellulose membranes were blotted with the indicated antibodies and developed as described above.

Immune Complex Enzyme Assays
Src activity was assayed in vitro with enolase as an exogenous substrate, essentially as previously described.⁸ In brief, Src was immunoprecipitated from stimulated RASM cells (100 nmol/L Ang II for 2 minutes). The immunoprecipitates were washed three times in lysis buffer and twice in kinase reaction buffer. Enolase as an exogenous Src substrate and CP-118,556 at 0.01 to 100 µmol/L was added before the start of the in vitro kinase reaction. The kinase reaction was performed for 10 minutes at 30°C and stopped by the addition of SDS sample buffer and boiling. Proteins were size-fractionated by SDS-PAGE. The gel was subjected to an autoradiography, and phosphorylation of enolase was quantified by densitometry in the linear range of film development using NIH Image 1.59. The density values in arbitrary units were plotted on a logarithmic scale, and IC₅₀ values were calculated using the curve-fit function of Cricket Graph (Cricket Software). An in vitro EGF receptor kinase assay using a synthetic tyrosine–containing peptide (Arg-Arg-Src) as exogenous substrate was performed as previously described.²⁰ In brief, purified membrane fractions of A431 cells, containing high amounts of EGF receptor kinase, were prepared.²⁰ Approximately 6 to 8 µg membrane protein, 2 mmol/L exogenous EGF receptor substrate Arg-Arg-Src, and 0.01 to 100 µmol/L CP-118,556 were added to the kinase reaction buffer. The reaction was initiated by the addition of 3 µmol/L EGF, allowed to proceed for 5 minutes at 30°C, and stopped by the addition of 3.3% trichloroacetic acid. Precipitated proteins were removed by centrifugation (14 000g for 5 minutes), and 50 µL of the supernatant, containing the synthetic peptide, was spotted onto phosphocellulose paper. After washing with 0.1% phosphoric acid, incorporation of [³²P]orthophosphate into Arg-Arg-Src was assayed by liquid scintillation counting. The results of the scintillation counting were plotted on a logarithmic scale, and the IC₅₀ value was determined as described above.

Measurement of Phosphotyrosine-Containing Proteins
For in vivo inhibition studies with CP-118,556, RASM cells were pretreated with CP-118,556 for 15 minutes and then stimulated with 40 ng/mL PDGF-BB for 10 minutes or 100 nmol/L Ang II for 5 minutes. Cell lysis and immunoprecipitation were performed as described above using a monoclonal antibody against the PDGF-ß receptor for PDGF-stimulated cells and against PLC- for Ang II–stimulated cells. Protein samples were size-fractionated by SDS-PAGE, and Western blotting with anti-phosphotyrosine antibody PY20 was performed. For quantification of PDGF-ß receptor and p97 tyrosine phosphorylation, films were scanned and densitometry was performed using NIH Image 1.59. The densitometry values in arbitrary units were plotted on a logarithmic scale, and IC₅₀ values were calculated as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Stimulates Phosphorylation of Several Proteins That Are Coprecipitated by PLC- Antibodies
To define proteins that interact with PLC- and may regulate PLC- activation by Ang II, RASM cells were labeled with [³²P]orthophosphate, and PLC- was immunoprecipitated. To provide additional information on these proteins, RASM cells were treated with 100 µmol/L sodium orthovanadate for 1 hour to inhibit protein tyrosine phosphatases and thereby increase tyrosine phosphorylation. In addition, PKC was downregulated by treating cells for 24 hours with 1 µmol/L PDBU, which has been shown to increase PLC activity.²¹ Stimulation of RASM cells by 100 nmol/L Ang II for 5 minutes led to increased phosphorylation of several proteins that coprecipitated with PLC- (Fig 1A). The most prominent PLC-–associated proteins had molecular masses of 97, 84, and 47 kD (p97, p84, and p47, respectively). The 145-kD protein was identified as PLC- by Western blotting (Fig 6C). When the film was exposed for longer times, increased phosphorylation of a 60-kD protein (p60) was detected (Fig 1B). Each of these proteins was then characterized by use of antibodies raised against proteins of similar molecular weights.

View larger version (46K): [in this window] [in a new window]   Figure 1. Ang II stimulates phosphorylation of several proteins that can be immunoprecipitated by PLC- antibodies. RASM cells were labeled with [32P]orthophosphate, as described in "Materials and Methods." Two dishes were pretreated with 100 µmol/L sodium orthovanadate for 1 hour (lanes 3 and 4), and one of these dishes was pretreated with 1 µmol/L PDBU for 24 hours (lane 4). RASM cells were stimulated with 100 nmol/L Ang II for 5 minutes (lanes 2 to 4), lysed in buffer containing 1% Triton X-100, immunoprecipitated with mixed monoclonal PLC- antibody, and analyzed by SDS-PAGE. The gel was dried and submitted to autoradiography for 1 hour (A) and 4 hours (B). Molecular masses of putative PLC-–associated phosphorylated proteins are indicated on the right. IP indicates immunoprecipitation. Results shown are representative of three independent experiments.

View larger version (77K): [in this window] [in a new window]   Figure 6. The [32P]orthophosphate-labeled 97-kD protein is identical to tyrosine-phosphorylated p97. RASM cells were labeled with [32P]orthophosphate as described in "Materials and Methods." RASM cells were stimulated with 100 nmol/L Ang II for 5 minutes, lysed in buffer containing 1% Triton X-100, immunoprecipitated with mixed monoclonal PLC- antibody, analyzed by SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was subjected to autoradiography (A) and then blotted with HRP-coupled anti-phosphotyrosine antibody PY20 (B) or PLC- antibody (C). IP indicates immunoprecipitation. Results shown are representative of three experiments.

Ang II Stimulates Phosphorylation of a 47-kD Protein in PLC- Immunoprecipitates: Identification as Nck
The 47-kD protein was identified as the adapter molecule Nck (Fig 2). Nck has been previously shown by others^{22 23} to be immunoprecipitated by PLC- antibodies, as demonstrated by the cross-reactivity of these antibodies with SH3 domains of Nck, which are highly homologous to PLC-. Lane 1 in Fig 2 (lane numbers, although they do not appear on figures, are assumed [left to right]) shows that PLC- antibodies recognize Nck on Western blots by virtue of this homology. Phosphorylation of Nck increased upon Ang II stimulation of RASM cells and was further enhanced by pretreating the cells for 1 hour with 100 µmol/L sodium orthovanadate (Fig 1A, lanes 2 and 3), whereas downregulation of PKC by PDBU pretreatment inhibited Nck phosphorylation (Fig 1A, lane 4). However, tyrosine phosphorylation of Nck could not be detected by Western blot analysis with anti-phosphotyrosine antibody (Fig 2, lanes 3 and 4). These findings suggest that Ang II stimulates a serine-threonine kinase in RASM cells that phosphorylates Nck. The activity of this serine-threonine kinase appears to be partially PKC dependent (Fig 1A, lane 4) and partially tyrosine kinase dependent (Fig 1A, lane 3).

View larger version (21K): [in this window] [in a new window]   Figure 2. Nck coprecipitates with PLC- but is not tyrosine-phosphorylated. Cell lysates of RASM cells were obtained as described in "Materials and Methods." Protein samples were immunoprecipitated with antibodies to Nck or PLC-, analyzed by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with Nck antibody, PLC- antibody, or anti-phosphotyrosine antibody 4G10 (pTyr). IgG detected by the secondary antibody (IgGH) is indicated. IP indicates immunoprecipitation.

Ang II Stimulates Phosphorylation of a 60-kD Protein in PLC- Immunoprecipitates: Identification as a Protein Other Than Ras-GAP p60
Ang II stimulation increased phosphorylation of a 60-kD protein that coprecipitated with PLC- (Fig 1B). Phosphorylation of this protein was increased slightly by pretreatment of RASM cells with 100 µmol/L sodium orthovanadate for 1 hour. PKC downregulation did not affect phosphorylation of the 60-kD protein. Association of a tyrosine-phosphorylated 60-kD protein (termed p60 in the present study) with PLC- has been reported for murine C3H10T1/2 fibroblasts transfected with v-Src¹¹ and for Jurkat T cells upon CD2 stimulation.²⁴ To determine whether the 60-kD protein in RASM cells is the PLC-–associated p60 described by Maa et al¹¹ and Hubert et al,²⁴ we performed anti-phosphotyrosine Western blots of PLC- immunoprecipitates of RASM cells. As shown in Fig 3, Ang II failed to stimulate tyrosine phosphorylation of a PLC-–associated 60-kD protein, whereas p60 was tyrosine-phosphorylated in v-Src–transformed NIH-3T3 cells. Immunoprecipitation of p60 with anti-p60 monoclonal antibody 2C4 from v-Src–transformed NIH-3T3 cells showed comigration with the PLC-–associated p60 of v-Src–transformed NIH-3T3 cells. Both the PLC-–associated p60 and the p60 protein immunoprecipitated by the 2C4 antibody from v-Src–transformed NIH-3T3 cells failed to react with antibody to c-Src (data not shown). Thus, the 60-kD PLC-–associated protein observed in Fig 1B is not the p60 that associates with PLC- in v-Src–transformed cells. p60 was also shown to be a substrate for the insulin receptor in CHO cells overexpressing the human insulin receptor.¹⁴ However, in RASM cells stimulated by 1 µmol/L insulin, tyrosine phosphorylation of a 60-kD protein could not be demonstrated (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 3. Ang II does not stimulate tyrosine phosphorylation of PLC-–associated p60. Cell lysates of RASM cells stimulated with 100 nmol/L Ang II for the indicated time points and of v-Src–transformed NIH-3T3 cells were obtained as described in "Materials and Methods." Protein samples were immunoprecipitated with mixed monoclonal PLC- antibody, monoclonal p60 antibody (2C4), or mouse preimmune serum (N), analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phosphotyrosine antibody 4G10 (pTyr). For immunoprecipitation (IP) with serum N, cell lysates from unstimulated RASM cells were used. TCL denotes total cell lysate (20 µg protein) from unstimulated RASM cells. Tyrosine-phosphorylated p60 is present in v-Src–transformed NIH-3T3 cells but not in Ang II–stimulated RASM cells. Results shown are representative of three independent experiments.

To characterize the 60-kD protein present in PLC- immunoprecipitates further, we studied proteins that associate with tyrosine-phosphorylated Ras-GAP.^{14 15 16} Ogawa et al¹⁵ showed a 60-kD protein associated with Ras-GAP in c-Src–transformed NIH-3T3 cells that comigrated with a tyrosine-phosphorylated 60-kD protein immunoprecipitated by the monoclonal antibody 2C4.¹⁵ Because Ang II stimulates Ras²⁵ and has been recently shown to stimulate tyrosine phosphorylation of Ras-GAP,²⁶ we determined whether p60 associated with Ras-GAP in Ang II–stimulated cells. Stimulation of RASM cells with 100 nmol/L Ang II led to a time-dependent increase in tyrosine phosphorylation of Ras-GAP that peaked at 1 minute (Fig 4A). However, there was no 60-kD protein whose tyrosine phosphorylation was stimulated by Ang II in Ras-GAP immunoprecipitates from Ang II–stimulated RASM cells (Fig 4B, lower blot, lanes 1 and 2). Analysis of Ras-GAP at 1 and 10 minutes also failed to demonstrate p60 (data not shown). As a positive control, we used v-Src–transformed NIH-3T3 cells, which exhibit increased tyrosine phosphorylation of many signaling molecules. v-Src–transformed NIH 3T3 cells readily showed tyrosine phosphorylation of a 60-kD protein that was present in Ras-GAP immunoprecipitates (Fig 4B, lower blot, lane 3). Using the 2C4 antibody to immunoprecipitate p60, we also demonstrated this finding (Fig 4B, lower blot, lane 6). However, no tyrosine-phosphorylated p60 was detected in 2C4 immunoprecipitates from Ang II–stimulated cells (Fig 4B, lower blot, lane 5). These findings indicate that p60 is not tyrosine-phosphorylated in RASM cells in response to Ang II.

View larger version (41K): [in this window] [in a new window]   Figure 4. Ang II stimulates tyrosine phosphorylation of monoclonal Ras-GAP antibody (Ras-GAP) but not p60. Cell lysates of RASM cells or of v-Src–transformed NIH-3T3 cells were obtained as described in "Materials and Methods." RASM cells were stimulated for the indicated times with 100 nmol/L Ang II. A, Protein samples were immunoprecipitated with Ras-GAP, transferred to nitrocellulose membranes, and blotted with anti-phosphotyrosine antibody 4G10 (pTyr). Ang II stimulated Ras-GAP tyrosine phosphorylation maximally at 1 minute. B, Protein samples were immunoprecipitated with Ras-GAP or with monoclonal p60 antibody (2C4), analyzed by SDS-PAGE, transferred to nitrocellulose membranes, and blotted either with Ras-GAP (upper blot) or with anti-phosphotyrosine antibody (lower blot). p60 in v-Src–transformed cells, but not RASM cells, associates with Ras-GAP and is tyrosine-phosphorylated. IP indicates immunoprecipitation. Results shown are representative of two independent experiments.

Ang II Stimulates Phosphorylation of 84- and 97-kD Proteins: Identification of p97 as a Tyrosine-Phosphorylated Protein
Ang II stimulated phosphorylation of 84- and 97-kD proteins in PLC- immunoprecipitates. Phosphorylation of these proteins was enhanced by pretreating the cells for 1 hour with 100 µmol/L sodium orthovanadate (Fig 1A). PKC downregulation of sodium orthovanadate–treated cells did not affect phosphorylation of p84 and p97 (Fig 1A). Anti-phosphotyrosine Western blots of PLC- immunoprecipitates showed a time-dependent increase in tyrosine phosphorylation of PLC-–associated p97 upon stimulation with 100 nmol/L Ang II, with maximal phosphorylation at 5 to 10 minutes (Fig 5). Anti-phosphotyrosine Western blots of PLC- immunoprecipitates failed to demonstrate an Ang II–stimulated increase in tyrosine phosphorylation of p84 (Fig 5). To prove that the 97-kD protein detected in [³²P]orthophosphate-labeled RASM cells (Fig 1) corresponds to the 97-kD phosphotyrosine protein detected by Western blotting, we labeled RASM cells with [³²P]orthophosphate, immunoprecipitated PLC-, size-fractionated the protein samples in an SDS gel, and transferred the proteins to a nitrocellulose membrane. The membrane was first subjected to autoradiography (Fig 6A) and subsequently blotted with anti-phosphotyrosine antibodies (Fig 6B). The PLC-–associated 97-kD protein labeled with [³²P]orthophosphate was identical to the 97-kD protein detected by anti-phosphotyrosine Western blotting, as measured by electrophoretic mobility. The increase in total phosphorylation stimulated by Ang II paralleled the increase in tyrosine phosphorylation (Fig 6A and 6B). Finally, equal amounts of PLC- were immunoprecipitated in both control and Ang II–treated cells (Fig 6C).

View larger version (20K): [in this window] [in a new window]   Figure 5. Ang II stimulates tyrosine phosphorylation of PLC-–associated p97, but not p84. A, Cell lysates of RASM cells stimulated with 100 nmol/L Ang II for the indicated time points were obtained as described in "Materials and Methods." Protein samples were immunoprecipitated with mixed monoclonal PLC- antibody, analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phosphotyrosine antibody 4G10 (pTyr). B, The relative increase of p97 tyrosine phosphorylation over control was determined by performing quantitative densitometry, as described in "Materials and Methods." IP indicates immunoprecipitation. Results shown are mean±SEM from at least three independent experiments for each time point.

p97 Tyrosine Phosphorylation Is Dependent on Src-Kinase Activity
Because p97 was the only PLC-–associated protein that was tyrosine-phosphorylated in an Ang II–dependent manner, we characterized signal events that may regulate its phosphorylation. To investigate the role of Src in p97 tyrosine phosphorylation, we first examined the effect of different tyrosine kinase inhibitors on p97 tyrosine phosphorylation in RASM cells. It has recently been shown that herbimycin A and tyrphostin 23 effectively block Ang II–mediated activation of PLC-.^{6 27} Therefore, we pretreated RASM cells with 1 µmol/L herbimycin A or 100 µmol/L tyrphostin 23 for 16 hours. After treatment with these inhibitors, Ang II stimulation of p97 tyrosine phosphorylation was completely inhibited (Fig 7A). Since herbimycin A and tyrphostin 23 are known to inhibit several receptor and nonreceptor tyrosine kinases, we investigated the effect of the recently described tyrosine kinase inhibitor CP-118,556, which has been reported to preferentially inhibit Src-family tyrosine kinases.¹⁷ To test for preferential inhibition of Src over other tyrosine kinases in RASM cells, we performed several in vitro and in vivo inhibition studies with CP-118,556 and determined IC₅₀ values for different tyrosine kinases. In vitro inhibition of activated Src by CP-118,556, immunoprecipitated from Ang II–stimulated RASM cells, yielded an IC₅₀ of 1.32±0.27 µmol/L, whereas in vitro inhibition of stimulated EGF receptor kinase yielded an IC₅₀ of 14.9 µmol/L. The IC₅₀ for in vivo inhibition of PDGF-ß receptor tyrosine phosphorylation was 8 µmol/L. Pretreatment of RASM cells with CP-118,556 for 15 minutes inhibited Ang II–stimulated p97 tyrosine phosphorylation in a concentration-dependent manner (Fig 7B), yielding an IC₅₀ of 1.4± 0.7 µmol/L. Thus, the IC₅₀ that we determined for activated c-Src in RASM cells correlated well with the IC₅₀ for p97 tyrosine phosphorylation, indicating that in fact c-Src was the relevant kinase for p97 tyrosine phosphorylation. However, the IC₅₀ values that we determined for EGF receptor kinase and PDGF-ß receptor kinase were only 10.7-fold and 6-fold higher, respectively, than the IC₅₀ for c-Src. Furthermore, the IC₅₀ of 1.3 µmol/L that we determined for c-Src in RASM cells was considerably higher than the IC₅₀ of 170 nmol/L previously reported by Hanke et al.¹⁷ Therefore, we could not exclude nonspecific inhibition by CP-118,556 of other tyrosine kinases that were stimulated by Ang II in RASM cells and might have contributed to p97 tyrosine phosphorylation.

View larger version (26K): [in this window] [in a new window]   Figure 7. Tyrosine phosphorylation of p97 is inhibited by tyrosine kinase inhibitors. RASM cells were pretreated with 1 µmol/L herbimycin A (HerbA), 100 µmol/L tyrphostin 23 (Tyr 23), or vehicle (dimethyl sulfoxide [DMSO]) for 16 hours (A) or with the Src-family tyrosine kinase inhibitor CP-118,556 at the indicated concentrations for 15 minutes (B). Cells were then harvested (time=0) or stimulated with 100 nmol/L Ang II for 5 minutes, and cell lysates were prepared as described in "Materials and Methods." Protein samples were immunoprecipitated with mixed monoclonal PLC- antibody, analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phosphotyrosine antibody 4G10 (pTyr). IP indicates immunoprecipitation. Results shown are representative of two independent experiments.

To address the role of Src in p97 tyrosine phosphorylation more specifically, we performed additional experiments in cells in which c-Src had been deleted by targeted gene disruption and in cells overexpressing v-Src. MASM cells from Src-/- mice and the corresponding wild-type mice¹⁸ as well as v-Src–transformed NIH-3T3 cells and the corresponding parent NIH-3T3 cells¹⁹ were prepared. Immunoprecipitation of PLC- from MASM and NIH-3T3 cells showed comigration of a PLC-–associated tyrosine-phosphorylated 97-kD protein with p97 from RASM cells (data not shown). Ang II stimulated tyrosine phosphorylation of p97 by 1.9±0.6-fold (mean±SEM, n=3) in wild-type MASM cells, whereas in Src-/- MASM cells there was no significant increase in p97 tyrosine phosphorylation (0.8±0.2-fold increase, n=3, Fig 8). The increase in p97 tyrosine phosphorylation in wild-type MASM cells corresponded to the 2-fold increase in c-Src activity upon Ang II stimulation of these cells (M. Ishida, unpublished data, 1996). Conversely, PLC-–associated p97 tyrosine phosphorylation was enhanced in v-Src–transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells (Fig 9). Several tyrosine-phosphorylated proteins that associated with PLC- in v-Src–transformed NIH-3T3 cells (Fig 9) could not be detected in RASM or MASM cells. In summary, p97 tyrosine phosphorylation appears to be dependent on c-Src activity, as shown by decreased tyrosine phosphorylation after treatment with Src-family tyrosine kinase inhibitors, increased phosphorylation in cells overexpressing v-Src, and decreased phosphorylation in Src-/- cells.

View larger version (19K): [in this window] [in a new window]   Figure 8. p97 tyrosine phosphorylation is diminished in Src-/- MASM cells. A, Cell lysates of wild-type (WT) or Src-/- MASM cells either left untreated or stimulated with 100 nmol/L Ang II for 5 minutes were obtained as described in "Materials and Methods." Protein samples were immunoprecipitated with mixed monoclonal PLC- antibody, analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phosphotyrosine antibody 4G10 (pTyr). B, The relative increase of p97 tyrosine phosphorylation over control in WT and Src-/- MASM cells was determined by quantitative densitometry as described in "Materials and Methods." IP indicates immunoprecipitation. Results shown are mean±SEM from three independent experiments.

View larger version (38K): [in this window] [in a new window]   Figure 9. Tyrosine phosphorylation of p97 is increased in v-Src–transformed NIH-3T3 cells. Cell lysates of v-Src–transformed NIH-3T3 cells (v-Src) or nontransformed NIH-3T3 cells (wild-type [WT]) were obtained as described in "Materials and Methods." Protein samples were immunoprecipitated with mixed monoclonal PLC- antibody, analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-phosphotyrosine antibody 4G10 (pTyr). Tyrosine-phosphorylated PLC-, p97, and p60 are denoted by arrows. Additional tyrosine-phosphorylated proteins in v-Src NIH-3T3 cells that were not found in RASM cells are denoted by asterisks. IgG detected by the secondary antibody is indicated. IP indicates immunoprecipitation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major finding of the present study is that Ang II stimulates phosphorylation of several PLC-–associated proteins, one of which is a tyrosine-phosphorylated 97-kD protein. Tyrosine phosphorylation of p97 appeared to depend on Src activity, on the basis of results with preferential Src-family tyrosine kinase inhibitors, Src-/- cells, and v-Src–overexpressing cells. Thus, p97 is a putative Src substrate that interacts with PLC- and may serve as a linker between c-Src and PLC-. Characterization of the other PLC-–associated proteins revealed Nck, which we believe is not physiologically relevant, since its coprecipitation is a result of PLC- antibodies recognizing homologous SH3 domains. An 84-kD protein was associated with PLC- and phosphorylated in response to Ang II, but it was not tyrosine-phosphorylated. Finally, a 60-kD protein was associated with PLC- and phosphorylated in response to Ang II. Characterization of this protein revealed that it was not the p60 shown to associate with PLC- and Ras-GAP.^{11 14 15 16 24}

PLC- activity is stimulated after phosphorylation²⁸ both by receptor tyrosine kinases, such as the PDGF receptor, and by nonreceptor tyrosine kinases, such as the Src family, ZAP-70, and Syk kinases.^{9 29 30 31} Activation of PLC- by the PDGF receptor requires PLC- binding to the receptor, which is mediated through PLC- SH2 domains.^{32 33} A model for activation of PLC- by nonreceptor tyrosine kinases has been recently proposed for B-cell receptor signaling.²⁹ In this model, binding of ligand to the B-cell receptor causes receptor clustering, which induces Syk to associate with the B-cell receptor. Syk then autophosphorylates, creating docking sites for PLC- SH2 domains on Syk. After PLC- binds to Syk, PLC- is tyrosine-phosphorylated and activated by Syk. We have established previously that c-Src activation is critical for Ang II–mediated stimulation of PLC- in RASM cells.^{6 7 8} However, our present results, as well as reports from other laboratories,^{10 34} suggest that c-Src does not directly activate PLC-. Specifically, we failed to show a direct association between PLC- and c-Src. This finding is in agreement with studies of Morrison et al,¹⁰ who could not detect association of mammalian PLC- with baculovirus-expressed c-Src in vitro. Pleiman et al³⁴ demonstrated that the PLC-1 immunoprecipitates from HeLa cells overexpressing c-Src did not contain c-Src, whereas association of PLC- with Fyn was observed. Finally, no one has shown binding of PLC-1 SH2 domains to c-Src in vitro or vice versa.

We hypothesized that a linker protein, specifically p60,^{11 12 13 15 24} mediates the interaction between PLC- and c-Src. p60 was shown to be a c-Src substrate in vitro¹⁵ and an in vivo substrate of v-Src, since it is tyrosine-phosphorylated in v-Src–transformed cells.¹² In v-Src–transformed murine fibroblasts, p60 associates with PLC-.¹¹ Finally, tyrosine phosphorylation of p60 was inhibited by the activation of PKC- in CHO cells overexpressing different members of the insulin receptor family.³⁵ The latter finding is important to our hypothesis, because Brock et al²¹ found that downregulation of PKC in RASM cells enhanced Ang II–stimulated PLC activity, suggesting a role for p60 in Ang II signal transduction. The failure to detect p60 tyrosine phosphorylation after Ang II stimulation of RASM cells or to immunoprecipitate tyrosine-phosphorylated p60 from cell lysates implies that this protein is not expressed in vascular smooth muscle. However, because the 2C4 antibody used in the present study cannot be used for Western blotting and since p60 has not been cloned to date, its presence in vascular smooth muscle cannot be excluded.

Two of the PLC-–associated proteins, p84 and p97, are candidates to mediate interactions between PLC- and c-Src, on the basis of findings that Ang II increases their phosphorylation. As shown by others,^{22 23} several of the PLC- antibodies cross-react with the adapter molecule Nck, leading to coimmunoprecipitation of Nck. Thus, the phosphorylated proteins present in PLC- immunoprecipitates could be associated with either PLC- or Nck. Several lines of evidence suggest that p84 and p97 are complexed to PLC- rather than Nck. First, when we immunoprecipitated Nck with an Nck-specific antibody that did not cross-react with PLC-, we did not coprecipitate p84 or p97 (data not shown). Second, Meisenhelder and Hunter²² found that Nck did not complex with other molecules under cell lysis conditions similar to those used in our experiments. Finally, Park and Rhee²³ did not observe a p84 that was coprecipitated with Nck antibodies.

The most interesting PLC-–associated protein in RASM cells was a 97-kD protein, which was tyrosine-phosphorylated upon Ang II stimulation in an Src-dependent manner. PLC-–associated p97 has been described previously in several reports.^{22 23 30 36 37 38} Increased phosphorylation of p97 in several cell systems has been demonstrated in response to multiple stimuli, including PDGF, phorbol ester, and vascular endothelial growth factor. For example, stimulation of NIH-3T3 cells by PDGF,³⁰ activation of platelets by thrombin,³⁸ treatment of A431 cells with phorbol ester or forskolin,²³ stimulation of bovine aortic endothelial cells with vascular endothelial growth factor,³⁶ and perfusion of liver with H₂O₂ and sodium orthovanadate³⁷ were reported to increase tyrosine phosphorylation of p97. To date, there has been no functional characterization of p97. The present study is the first to demonstrate that Src activity is required for p97 tyrosine phosphorylation. These data suggest that p97 may participate in Ang II–induced signal transduction events that link c-Src activation to stimulation of PLC-.

Finally, the present results indicate that Ang II stimulates phosphorylation of Nck. Nck contains three SH3 domains and one SH2 domain and belongs to the class of adapter molecules that includes Grb2, Crk-I, and Crk-II.^{39 40 41} This is the first study to demonstrate that a G protein–coupled receptor stimulates phosphorylation of Nck. However, the function of Nck phosphorylation in Ang II signal transduction remains to be elucidated.

In summary, Ang II stimulation of RASM cells increases phosphorylation of several PLC-–associated proteins that may act as mediators of PLC- activation. Tyrosine-phosphorylated p60, a putative adapter protein for c-Src and PLC-, was not present in RASM cell PLC- immunoprecipitates. Instead, a tyrosine-phosphorylated p97 protein was identified that appeared to be a candidate to mediate PLC- activation. This protein associated with PLC-, exhibited increased tyrosine phosphorylation in response to Ang II, and was dependent on Src activity for its phosphorylation. Future studies to identify this p97 protein will be required to define its physiological role in PLC- activation and Ang II signal transduction.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1 receptor = Ang II type-1 receptor CHO = Chinese hamster ovary ECL = enhanced chemiluminescence EGF = epidermal growth factor GAP = GTPase-activating protein HRP = horseradish peroxidase MASM = mouse aortic smooth muscle PDBU = phorbol 12,13-dibutyrate PDGF = platelet-derived growth factor PIP2 = phosphatidylinositol 4,5-bisphosphate PKC = protein kinase C PLC = phospholipase C PMSF = phenylmethylsulfonyl fluoride RASM = rat aortic smooth muscle Src-/- = c-Src knockout VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (HL-44721 and HL-49192 to Dr Berk) and the Deutsche Forschungsgemeinschaft (SCHM 1174/1-1 to Dr Schmitz). Dr Berk is an Established Investigator of the American Heart Association. We thank members of the Berk laboratory for their help.

	Footnotes

 
This manuscript was sent to Laurence H. Kedes, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received October 25, 1996; accepted May 14, 1997.

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