©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin Stimulates the Tyrosine Dephosphorylation of pp125 Focal Adhesion Kinase (*)

(Received for publication, November 3, 1994)

Tahir S. Pillay (§) Toshiyasu Sasaoka (¶) Jerrold M. Olefsky (**)

From the Division of Endocrinology and Metabolism, Department of Medicine, University of California San Diego, La Jolla, California 92093-0673 and the Research Service, Veterans Administration Medical Center, San Diego, California 92161

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The phosphorylation state of pp125 focal adhesion kinase in response to insulin was examined in parental and transfected Rat-1 fibroblasts expressing both wild-type (HIRc cells) and mutant human insulin receptor cDNAs lacking the C-terminal twin tyrosine phosphorylation sites (YF2 cells) or a deletion mutant lacking the distal 43 amino acids of the beta-subunit (DeltaCT cells). In HIRc cells insulin stimulated the tyrosine dephosphorylation of pp125, whereas IGF-I did not. In contrast, the tyrosine phosphorylation state of pp125 was unchanged in the parental Rat-1 fibroblasts and the YF2 or DeltaCT mutant cell lines in response to insulin. Analysis of the supernatants revealed that pp125 was only one component of the major M(r) 120-130-kDa phosphotyrosine band seen in HIRc cells. We conclude that: 1) in contrast to other growth factors, insulin stimulates the dephosphorylation of pp125; 2) the presence of the insulin receptor C-terminal tyrosines 1328 and 1334 is required for the insulin-stimulated tyrosine dephosphorylation of pp125, suggesting a possible SH2 domain-dependent interaction; 3) insulin may modulate integrin-mediated signaling through pp125 by altering the phosphorylation state of pp125.


INTRODUCTION

Insulin stimulates autophosphorylation of its receptor, resulting in activation of the intrinsic tyrosine kinase activity of the receptor and the phosphorylation of its major intracellular substrates leading to signal transduction(1, 2) . A number of substrates have been identified, of which IRS-1 and SHC have been the most extensively characterized(1, 2, 3) . Phosphorylation of these molecules, particularly IRS-1, in response to insulin enables the recruitment of numerous secondary signaling molecules that contain SH2 domains. These include the p85 subunit of phosphatidylinositol 3-kinase, GRB2, and the SH2 domain containing phosphatase Syp (SHPTP2, PTP1D, PTP2C)(1, 2) .

Much of the research on insulin receptor signaling has focused on the identification of proteins that undergo increases in tyrosine phosphorylation as a result of receptor activation. In comparison, relatively little attention has been paid to the role of tyrosine dephosphorylation in insulin action. Protein tyrosine phosphatases may act as transducers or modulators of signaling pathways. Recently, we and others (4, 5) have shown that inhibition of the tyrosine phosphatase Syp blocks insulin stimulation of mitogenesis and MAP kinase activity, indicating that Syp-mediated tyrosine dephosphorylation positively participates in the signal transduction cascade leading to cell growth.

The purpose of this study was to identify phosphoproteins that undergo tyrosine dephosphorylation in response to insulin. This led to the identification of pp125 as a target protein for insulin-stimulated tyrosine dephosphorylation.


EXPERIMENTAL PROCEDURES

Cell Culture and Materials

The cell lines used were Rat-1 fibroblasts stably transfected with and overexpressing both wild-type human insulin receptor (HIRcB) and mutant receptors lacking either the C-terminal tyrosine phosphorylation sites or the distal 43 amino acids of the beta-subunit. These were cultured as described previously(6, 7, 8) . Porcine insulin was obtained from Lilly. A mouse monoclonal antibody to pp125 focal adhesion kinase (clone 2A7) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Enhanced chemiluminescence detection reagents were purchased from Amersham Corp. Nitrocellulose was obtained from Schleicher and Schuell.

Immunoblotting

Cells were routinely cultured in 35-mm 6-well dishes as described and used at subconfluence. Approximately 16-24 h prior to experimental manipulations, the serum-containing medium was removed and replaced with fresh serum-free Hepes-buffered Dulbecco's modified Eagle's medium (Sigma) containing 1 mg/ml radioimmunoassay grade BSA (^1)(Sigma) for 16 h. The cells were then stimulated with various ligands as indicated. The incubation was terminated by rapid aspiration of the medium followed by the addition of 100 µl of Triton X-100 lysis buffer containing phosphatase and protease inhibitors(9) . After a 10-min incubation, the lysates were clarified by centrifugation at 10,000 times g, and pp125 was immunoprecipitated using 2-4 µg of monoclonal anti-pp125 (2A7) overnight followed by the addition of anti-mouse agarose for 1 h. The bound immunoprecipitates were washed in RIPA buffer (3 times 1 ml) and lysis buffer (2 times 1 ml) and then once in 100 mM Tris, pH 6.8 (1 ml). The pellets were boiled in Laemmli sample buffer containing 5% mercaptoethanol, and eluates were electrophoresed on 7.5% T/3% C Tricine/SDS-polyacrylamide gels. Proteins were transferred to 0.45-µm nitrocellulose using a Bio-Rad SD Transblot. The membrane was blocked for 1 h in 3% BSA in Tris-buffered saline (50 mM Tris, 150 mM NaCl, pH 7.4, with 0.1% Tween 20) (buffer A). The blocked membranes were probed with affinity-purified rabbit anti-phosphotyrosine antibody (9) (0.5-1 µg/ml) in Tris-buffered saline containing 0.1% BSA and 0.1% Tween 20 for 12 h, washed in Tris-buffered saline, 0.1% Tween 20, 1 mM EDTA (4 times 100 ml/10 min each), and then probed with anti-rabbit peroxidase conjugate (Amersham, 1/1000) for 1 h. The membranes were then washed extensively in buffer B as described above. Bound anti-phosphotyrosine antibody was detected using anti-rabbit peroxidase and the ECL reagent according to the manufacturer's instructions and autoluminography on preflashed Kodak X-Omat AR film. Band intensities on the autoluminographs were quantified by densitometry on a Hewlett-Packard ScanJet II using Scananalysis software (Elsevier Biosoft).


RESULTS

Insulin-stimulated Tyrosine Dephosphorylation of an M(r)120-130-kDa Protein

In an effort to identify tyrosine phosphoproteins dephosphorylated in response to insulin, HIRc cells were stimulated with insulin, followed by immunoblotting of whole cell lysates with an anti-phosphotyrosine antibody. As can be seen, a prominent M(r) 120-kDa protein underwent tyrosine dephosphorylation in a dose (Fig. 1A) and time (Fig. 1B) dependent fashion. Dephosphorylation was apparent at 10 min and maximal at 20 min (Fig. 1B).


Figure 1: Insulin-stimulated tyrosine dephosphorylation of an M(r) 120-130-kDa protein in HIRc cells. Whole cell lysates of HIRc cells stimulated with insulin were analyzed by anti-phosphotyrosine immunoblotting as described under ``Experimental Procedures.'' PanelA, dose response of insulin-stimulated dephosphorylation of the M(r) 120-130-kDa proteins. Serum-starved HIRc cells were stimulated with varying concentrations of insulin for 10 min as indicated. Immunoblots were developed using anti-rabbit-HRP, luminol, and chemiluminography. PanelB shows a time course of the insulin-stimulated dephosphorylation of the M(r) 120-130-kDa proteins.



Dephosphorylation of p125 Focal Adhesion Kinase

For a protein to be dephosphorylated on phosphotyrosine residues would require prior phosphorylation by a tyrosine kinase. Several proteins in the M(r) 120-130-kDa range have been characterized as potential tyrosine kinase substrates. These include pp125 and pp120, which are both substrates for pp60(10, 11, 12, 13) . In addition, pp125 undergoes tyrosine autophosphorylation in response to the binding of fibronectin to its receptor. In order to assess which protein(s) within the 120-130-kDa region displayed insulin-induced tyrosine dephosphorylation, we immunoprecipitated pp125 from lysates of insulin-stimulated cells and then immunoblotted the precipitates with anti-phosphotyrosine antibodies (Fig. 2). These experiments showed that pp125 was markedly dephosphorylated approximately 50% in response to insulin. Analysis of the supernatants revealed no tyrosine dephosphorylation of the remaining M(r) 120-130-kDa proteins, indicating that among the phosphoproteins migrating at 120-130 kDa, only pp125 exhibits insulin-induced tyrosine dephosphorylation. Furthermore, IGF-1 did not stimulate dephosphorylation of the M(r) 120-130-kDa proteins (Fig. 2), suggesting that pp125 dephosphorylation was relatively specific for the insulin signaling process.


Figure 2: Effects of IGF-1 on the phosphorylation of pp125 and other M(r) 120-130-kDa proteins. Serum-starved HIRc cells were exposed to insulin and IGF-1 as indicated for 15 min. pp125 was precipitated using antibody 2A7, and the immunoprecipitates (leftpanel) and supernatants (rightpanel) were analyzed by anti-phosphotyrosine immunoblotting and autoluminography as described under ``Experimental Procedures.''



An Intact Insulin Receptor C Terminus Is Required for pp125 Dephosphorylation

We next attempted to identify the mechanism whereby insulin stimulates the dephosphorylation of pp125. Autophosphorylation of the insulin receptor, particularly at the C-terminal twin tyrosines 1328 and 1334(14) , allows it to bind to the SH2 domain containing molecules, and we have recently shown that the SH2 domain of Syp can associate with the insulin receptor C terminus(15) . To assess the role of the C-terminal tyrosine residues, we conducted similar experiments as in Fig. 1and Fig. 2in DeltaCT and YF2 cells. We found that in parental Rat-1 fibroblasts and in DeltaCT and YF2 cells, which both lack the C-terminal residues(6, 7, 8) , insulin stimulation of pp125 dephosphorylation was not observed (Fig. 3), suggesting that phosphorylation of the distal C terminus is necessary for this process to occur.


Figure 3: Insulin-stimulated dephosphorylation of pp125 in transfected and untransfected Rat-1 fibroblasts. The cell lines indicated were stimulated with 0.1 µM insulin for 10 min, and p125 focal adhesion kinase was immunoprecipitated using antibody 2A7 as described under ``Experimental Procedures.'' The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with anti-phosphotyrosine antibodies. An autoluminograph is shown. HirC, Rat-1 cells transfected with wild-type human insulin receptor; Rat1, parental Rat-1 fibroblasts; YF2, cells transfected with insulin receptor lacking the C-terminal tyrosine phosphorylation sites; DeltaCT, Rat-1 cells transfected with a deletion mutant lacking the C-terminal 43 amino acids of the beta-subunit.




DISCUSSION

Activation of the insulin receptor after ligand binding results in the tyrosine phosphorylation of multiple endogenous substrates, and much effort has been devoted to the characterization of such substrates (1, 2) . The major endogenous phosphoprotein observed after insulin stimulation is IRS-1, which undergoes phosphorylation on multiple residues and recruits other signaling molecules that contain SH2 domains(2) . Apart from the primary action of tyrosine phosphorylation, tyrosine dephosphorylation may also play a critical role in the propagation of the insulin signal. Such dephosphorylation could either inhibit signal transmission or play a positive transducing role. Recently, we and others have shown that the SH2 domain containing phosphatase Syp exerts a positive role in insulin-stimulated mitogenesis, indicating a necessary role for a tyrosine dephosphorylation event in signal transduction(4, 5) . Syp may be activated by binding to IRS-1 or the insulin receptor(15, 16) . Activation of Syp or other tyrosine phosphatases (17) would lead to the dephosphorylation of susceptible tyrosine phosphoproteins.

The M(r) 120-130-kDa phosphoprotein is the major tyrosine phosphoprotein found in quiescent serum-starved monolayer HIRc cells. We have demonstrated that a component of this protein band is subject to insulin-stimulated tyrosine dephosphorylation in a time and dose-dependent manner. Using specific antibodies(10, 18) , we have identified the relevant phosphoprotein within the 120-130-kDa region as pp125. Thus, immunoprecipitation studies showed that pp125 is tyrosine-phosphorylated in unstimulated cells, and insulin stimulation results in marked tyrosine dephosphorylation. The time course of pp125 dephosphorylation is delayed relative to insulin receptor kinase activation and is maximal 10 min after peak insulin receptor and IRS-1 tyrosine phosphorylation. After immunoprecipitation of pp125, analysis of the supernatants revealed that the other phosphoproteins in this region are not dephosphorylated in response to insulin. The decrease in the level of tyrosine phosphorylation of pp125 may result from the activation of a tyrosine phosphatase or possibly the inhibition of a tyrosine kinase in response to insulin.

pp125 was first identified as a phosphotyrosine protein in chicken embryo fibroblasts transformed with v-src(10) . Subsequent cDNA cloning revealed that pp125, which was a substrate for pp60, was itself a novel tyrosine kinase (12, 19, 20, 21, 22) that colocalizes with components of focal adhesions(23) , tensin, vinculin, and talin. pp125 differs from other receptor and nonreceptor tyrosine kinases in that it contains a conserved catalytic domain flanked by large N- and C-terminal domains that lack sequence similarity to other tyrosine kinases(11) . In addition, it does not contain any sequence determinants for membrane association. Overexpression of activated c-src results in a large increase in pp125 tyrosine phosphorylation, which is blocked by the expression of kinase negative c-src(11) . Hence, pp125 may contribute to transformation of cells by v-src. Dephosphorylation of pp125 would prevent complex formation with and activation of pp60.

Both IRS-1 and the insulin receptor bind to the SH2 domain of Syp and activate it in vitro(4, 15, 16) . Microinjection and transfection studies indicate a positive role for Syp in insulin-stimulated mitogenesis(4, 5) . Potentially, activation of Syp in response to insulin could result in the dephosphorylation of substrates including pp125. However, we were unable to demonstrate the presence of Syp in pp125 immunoprecipitates from HIR cells, and a Syp-GST fusion protein containing a Syp SH2 domain did not precipitate pp125, although these do not rule out a role for Syp in the dephosphorylation of pp125. Alternatively, or additionally, there may be another tyrosine phosphatase mediating this effect(17) . Overexpression of a dominant negative Syp protein increases tyrosine phosphorylation of a 120-kDa protein in response to insulin, which binds to the SH2 domain of Syp and is not related to pp125(5) .

In the cell lines overexpressing the C-terminal insulin receptor mutants, insulin-stimulated IRS-1 phosphorylation is intact, and hence Syp activation is intact(6, 7, 8) . Taken together, this raises the possibility that a distinct phosphotyrosine phosphatase exists, which is activated in response to insulin and requires the presence of an intact beta-subunit C terminus for activation. Alternatively, the association of the insulin receptor with Syp could provide a unique localization of Syp not provided by IRS-1, which would imply that both activation and cellular localization are important for the functional effects of Syp.

Recently, a cDNA for a new tyrosine phosphatase designated PTPD1 has been cloned(24) . PTPD1 has an M(r) of 130,000 and associates with and is phosphorylated by Src kinase(24) . Src kinase associates with pp125(13, 25) as well, suggesting that PTPD1 and pp125 may colocalize. Several stimuli lead to the phosphorylation of 120-130-kDa proteins(21) . These include: clustering of B1 integrins by cell adhesion(26, 27, 28) ; stimulation by vasopressin(29) , bombesin(30, 31) , platelet-derived growth factor (32) , endothelin(29, 33) , and lysophosphatidic acid(33, 34, 35, 36) , and transformation of cells by v-src(21, 24) . These stimuli result in the phosphorylation and activation of focal adhesion kinase(18, 22, 24, 28, 31, 37) . In contrast, we have demonstrated that pp125 is dephosphorylated in response to insulin. The functional significance of this observation is unclear but suggests that insulin may regulate the adhesive ability of cells and consequently modulate integrin-mediated cell adhesion and migration.

Although the biologic role of pp125 dephosphorylation remains unknown, the fact that DeltaCT and YF2 cells do not mediate pp125 dephosphorylation provides some clues. Both cell lines exhibit enhanced mitogenesis in response to insulin(6, 7, 8) . Although speculative, since insulin-stimulated mitogenesis is enhanced in DeltaCT cells and YF2 cells, it is possible that dephosphorylation of pp125 serves to restrain entry of cells into the growth cycle. This would tend to retard insulin's mitogenic effects, consistent with the fact that the stronger mitogenic growth factor IGF-1 did not cause pp125 dephosphorylation. It is also interesting to note that of the two C-terminal phosphorylation sites in the insulin receptor beta-subunit, only the tyrosine corresponding to tyrosine 1334 is conserved in the IGF-1 receptor, while tyrosine 1328 is replaced by a phenylalanine. This is the first demonstration of a signaling difference between the insulin and IGF-1 receptors. This difference may arise from the phosphorylation of tyrosine 1328 of the insulin receptor, which may lead to an interaction with an SH2 domain containing protein. Interestingly, tyrosine 1328 is located adjacent to serine 1327, which is a major site of phosphorylation by protein kinase C(38, 39) . Phosphorylation of serine 1327 could regulate the interaction of tyrosine 1328 with its putative target, a hypothesis that deserves further investigation.

In summary, we have demonstrated that when Rat-1 cells transfected with human insulin receptors are stimulated with insulin, pp125 focal adhesion kinase is dephosphorylated by about 50%. This process is specific for insulin, does not occur with IGF-I, and requires the presence of the C-terminal tyrosine phosphorylation sites of the insulin receptor beta-subunit. The exact sites on pp125 focal adhesion kinase, which are dephosphorylated in response to insulin, are unknown, although it has a number of consensus tyrosine phosphorylation sites for SH2 domain binding(40) .


FOOTNOTES

*
This work was supported in part by the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK33651, the Veterans Administration Medical Research Service, the Sankyo Diabetes Research Fund, and the Juvenile Diabetes Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Juvenile Diabetes Foundation international fellowship.

Present address: Toyama Medical and Pharmaceutical University, Toyama, Japan 930-01.

**
To whom correspondence should be addressed. Tel.: 619-534-6651; Fax: 619-534-6653.

(^1)
The abbreviations used are: BSA, bovine serum albumin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; IGF, insulin-like growth factor.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.