vß3 Integrins and Pyk2 Mediate Insulin-Like Growth Factor I Activation of Src and Mitogen-Activated Protein Kinase in 3T3-L1 Cells
Hiroko Sekimoto,
Jodi Eipper-Mains,
Sunthorn Pond-Tor and
Charlotte M. Boney
Department of Pediatrics, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903
Address all correspondence and requests for reprints to: Charlotte M. Boney, M.D., Rhode Island Hospital, Department of Pediatrics, 593 Eddy Street, MPS-2, Providence, Rhode Island 02903. E-mail: Charlotte_Boney{at}brown.edu.
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ABSTRACT
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IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to
v and ß3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that
vß3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to
vß3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the ß3 subunit, consistent with inside-out activation of
vß3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of
vß3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.
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INTRODUCTION
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IGF-I HAS BROAD biological action, including regulation of cellular proliferation, differentiation, and migration. The biological effects of IGF-I are mediated through its receptor tyrosine kinase, which phosphorylates specific substrates to activate downstream signaling pathways (1). We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the adaptor protein Shc and the downstream ERK-1 and -2 MAPK pathway (2, 3). IGF-I regulation of Shc and MAPK activity, but not IRS-1 phosphorylation, is mediated through the activation of Src family kinases (4). IGF-I activation of c-Src in 3T3-L1 preadipocytes is rapid and occurs at the membrane (5), but the mechanism of IGF-I activation of c-Src is unknown.
Src localized to membranes mediates the mitogenic effects of numerous growth factor receptors, including G protein-coupled receptors and receptor tyrosine kinases such as the epidermal growth factor and platelet-derived growth factor receptors (6). Direct activation of Src by the epidermal growth factor and platelet-derived growth factor receptors has been demonstrated(7, 8); however, indirect activation of Src by growth factor receptors is more common, particularly through activation of integrins (9, 10). Integrins comprise a large family of heterodimeric cell surface adhesion receptors that connect the extracellular matrix to the intracellular cytoskeleton via formation of multiprotein signaling complexes called focal adhesions. The role of Src family tyrosine kinases in focal adhesion complexes includes recruitment and activation of downstream signaling pathways, including the Shc-Ras-MAPK pathway (6, 11).
In addition to Src, integrin signaling is mediated by a family of focal adhesion nonreceptor tyrosine kinases (12). Focal adhesion kinase (FAK), the most widely expressed member of this proline-rich tyrosine kinase family, plays an important role in integrin-mediated anchorage-dependent proliferation and motility (13). Whereas the activation of FAK is dependent on cell adhesion, the other kinase in this family, calcium-dependent proline-rich tyrosine kinase-2 (Pyk2), is activated by numerous extracellular signals as well as intracellular calcium (14). Pyk2 is expressed mainly in nerve and hematopoietic cells; however, it has a role in hormone-induced integrin signaling in smooth muscle and adipocytes (15, 16). Pyk2 and FAK act with Src to mediate integrin-facilitated growth factor receptor activation of the MAPK pathway (14, 17).
There is mounting evidence that cross talk between growth factor receptors, including the IGF-I receptor, and integrins is an important signaling mechanism in cell proliferation, adhesion, and migration (18, 19). However, the components and their interaction involved in the IGF-I receptor-integrin cross talk, which regulate downstream signaling, are not well understood. Growth factor-stimulated integrin signaling is referred to as inside-out activation, i.e. intracellular signals generated from growth factor receptors modulate cytoplasmic interactions of the integrin subunits, leading to a conformation change, increased extracellular binding of integrin ligands, and increased integrin signal transduction (20, 21, 22). Although studies have shown that
vß3 integrins regulate IGF-I receptor activation and mediate IGF-I action in smooth muscle cells (23, 24, 25, 26), there are few studies that address the role of integrins in IGF-I action in other cell types or the mechanism of integrin involvement in IGF-I mitogenic signaling. We have been studying the role of IGF-I in the proliferation of 3T3-L1 cells as a model of preadipocyte growth and differentiation to better understand hormonal regulation of adipogenesis. Other studies have shown that lipid rafts/caveolae are essential for IGF-I signaling in 3T3-L1 preadipocytes (27), suggesting integrin involvement in IGF-I action in 3T3-L1 cells. We hypothesized that IGF-I activation of Src and the downstream MAPK pathway in 3T3-L1 cells is mediated through integrin activation of Src-containing focal adhesion signaling complexes. We have determined that a multiprotein signaling complex consisting of
vß3 integrins, Src, and the FAK Pyk2 regulates IGF-I mitogenic signaling in 3T3-L1 cells.
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RESULTS
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vß3 Integrin Activation by IGF-I
There have been a number of reports demonstrating a role for integrins in IGF-I signaling, usually involving either
vß3 in smooth muscle (19) and trophoblasts (28) or
5ß1 in chondrocytes (29). We have previously shown that 10 nM IGF-I robustly activates MAPK in 3T3-L1 cells (2) and that Src is required for this activation (4), so we pretreated cells overnight with the integrin antagonist echistatin, a disintegrin that inhibits ß1 and ß3 subunits. Figure 1
shows IGF-I stimulation significantly increased MAPK activation 3- to 4-fold and increased Src phosphorylation by approximately 40%. The IGF-I-stimulated Src activation is modest compared with our previous reports because Src was immunoprecipitated from less total cell lysate protein and total cell lysates contain only a small fraction of Src that is membrane-associated and responsive to IGF-I. Echistatin (0.1 µM) dramatically inhibited basal as well as IGF-I-stimulated MAPK activity (Fig. 1A
). In addition, echistatin also inhibited IGF-I phosphorylation of Src (Fig. 1B
). To determine the identity of the integrin involved in IGF-I signaling to MAPK, we incubated cells with 10 µg/ml monoclonal blocking antibodies to different integrin subunits 2 h before addition of IGF-I. Blocking antibodies to ß3 and
v but not control IgG, anti-ß1 (Fig. 2
) or anti-
5 (data not shown) inhibited IGF-I activation of MAPK. Similar to the echistatin effect, blocking antibodies to ß3 and
v also inhibited basal MAPK activity.

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Fig. 1. Integrin Inhibition Impairs IGF-I Activation of MAPK and Src
A, Serum-starved subconfluent 3T3-L1 cells were treated with the disintegrin 0.1 µM echistatin overnight before stimulation with 10 nM IGF-I for 5 min for MAPK analysis and 1 min for Src analysis. Cell lysate proteins were analyzed by Western blotting using phospho-MAPK antibodies (P-MAPK) followed by ERK-1/2 antibodies (total MAPK). Representative blot is shown on the left, and densitometry of mean + SEM (n = 4; **, P < 0.0001) is shown on the right. B, Src was immunoprecipitated from cell lysates and analyzed by Western blotting using anti-PY418-Src antibodies followed by c-Src antibodies (total Src). Representative blot is shown on the left, and densitometry of mean + SEM (n = 4; *, P = 0.04) is shown on the right.
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Fig. 2. Blocking Antibodies to vß3 Inhibit IGF-I Activation of MAPK
Serum-starved subconfluent 3T3-L1 cells were treated with 10 µg/ml blocking antibodies to various integrin subunits or nonspecific mouse IgG (Control) for 2 h before stimulation with 10 nM IGF-I for 5 min. Cell lysates were analyzed by Western blotting using antibodies for phospho-MAPK (P-MAPK) and ERK-1/2 MAPK (total MAPK).
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These data suggest a role for
vß3 integrins in IGF-I mitogenic signaling, so we evaluated direct effects of IGF-I on integrin function by immunocytochemistry using a specific antibody that recognizes activation-dependent or ligand-induced binding site (LIBS) epitopes in ß3 integrin extracellular domains (20, 30, 31). Figure 3A
shows increased staining with LIBS antibody after stimulation of attached cells with 10 nM IGF-I for 1 min. Essentially undetectable staining was observed if the LIBS antibody was omitted or if nonspecific mouse IgG was used instead of anti-LIBS (data not shown). Ligand occupancy and integrin activation also result in tyrosine phosphorylation of the cytoplasmic tail of the ß3 subunit, which is important in signaling complex formation (25, 32, 33). Using phosphospecific antibodies to tyrosine 773 (sometimes referred to as tyrosine 747), we found an increase in ß3 phosphotyrosine by Western blot of cell lysates after stimulation of cells with IGF-I for 12 min (Fig. 3B
). The phosphorylated band migrated at the expected molecular mass (130 kDa) of the ß3 subunit. Blots were stripped and Western blotting for GAPDH demonstrated equal protein amounts per lane (data not shown). We were unable to detect murine ß3 protein in 3T3-L1 cell lysates by Western blotting or immunoprecipitate ß3 from cell lysates with commercially available antihuman ß3 antibodies. However, it is unlikely that treatment with IGF-I for 1 min would alter ß3 content. These results are direct evidence of IGF-I-mediated inside-out activation of
vß3 integrins.

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Fig. 3. IGF-I Stimulates vß3 Ligand Binding and ß3 Subunit Phosphorylation
A, Representative photomicrographs are shown of immunocytochemical analysis of specific ß3 LIBS epitope staining. Subconfluent 3T3-L1 monolayers were serum starved overnight and treated with or without 10 nM IGF-I for 1 min. Cells were fixed in 100% methanol, blocked in 10% normal goat serum and then incubated with 1:100 dilution of anti-LIBS in 10% goat serum. Secondary antibody was goat antimouse Alexa-Fluor, and coverslips were mounted with Vectashield plus 4',6-diamidino-2-phenylindole. B, Western blot analysis of 25 µg cell lysate protein from subconfluent cells treated with or without 10 nM IGF-I for 15 min is shown using anti-PY773-ß3 antibodies. Blots were cut and the lower half was exposed to anti-GAPDH antibodies to demonstrate equal protein in samples (not shown). Densitometry analysis of fold increase above basal (mean + SD, n = 4; *, P = 0.03 at 1 min, P = 0.04 at 2 min) is shown.
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Activation of FAK Signaling Kinases by IGF-I
Integrin activation results in recruitment of specific cytoskeletal proteins, integrin clustering, and focal adhesion formation (12). We investigated specific signaling molecules regulated by integrin activation and clustering, specifically the nonreceptor tyrosine kinase family of FAK and the related proline-rich tyrosine kinase Pyk2. In attached cells, FAK colocalizes with activated integrins, autophosphorylates at tyrosine 397, and then activates downstream signaling pathways to include MAPK (13). Phosphorylation at this site is known to regulate the binding of Src family kinase members as well as other SH2 domain-containing signaling proteins (13). Pyk2 is translocated to integrin focal adhesions (34) and contains an autophosphorylation site at tyrosine 402 (Y402), which binds Src and activates the MAPK pathway. We immunoprecipitated FAK and Pyk2 from proliferating 3T3-L1 cells treated with IGF-I for 1 min, then analyzed tyrosine phosphorylation by Western blotting (Fig. 4
). We saw a modest increase of approximately 30% in FAK PY-397, but a marked increase in phosphorylation of Pyk2 Y402, indicating significant Pyk2 activation (14). Both tyrosine kinases have been shown to bind Src in integrin signaling complexes, so we probed for coimmunoprecipitation of c-Src. Although Src was found in association with FAK, we saw no appreciable increase with IGF-I; however, Src was only found to associate with phosphorylated Pyk2 from IGF-I-treated cells.

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Fig. 4. Phosphorylation and Association of FAKs with Src in Response to IGF-I
FAK and Pyk2 were immunoprecipitated (IP) from subconfluent, serum-starved attached cells stimulated with 10 nM IGF-I for 1 min and analyzed by Western blotting. FAK blots were cut in half, and the upper half exposed to anti-PY397 antibodies followed by anti-FAK antibodies. Pyk2 blots were also cut and the upper half exposed to anti-PY402-Pyk2 antibodies followed by anti-Pyk2 antibodies. The lower half of the blots were analyzed using anti-Src antibodies. This experiment was repeated once with identical results.
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Integrin-facilitated activation of Pyk2 leads to the recruitment of Src family kinases and activation of the downstream MAPK pathway (34). Pyk2 activation is mostly Src independent (14, 35), and the association of Pyk2 PY402 with Src SH2 domains leads to Src activation (17). However, in some cells, Src activates Pyk2 through phosphorylation of multiple tyrosines (36, 37, 38). To evaluate the IGF-I-dependent activation of Pyk2 and Src, we treated cells for 30 min with the selective Src family kinase inhibitor PP1 (39) before stimulation with IGF-I and analyzed total Pyk2 phosphorylation. Figure 5
shows a representative Western blot of phosphorylated Pyk2 using antiphosphotyrosine 4G10 antibody. No significant effect of PP1 on IGF-I-stimulated tyrosine phosphorylation of Pyk2 was observed, but PP1 was very effective at blocking IGF-I-stimulated MAPK activity downstream of Src, as previously shown (4). We conclude from these data that IGF-I-stimulated Pyk2 phosphorylation is independent of Src kinase activity in 3T3-L1 cells.

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Fig. 5. Src Inhibition Has No Effect on IGF-I Phosphorylation of Pyk2
Serum-starved subconfluent 3T3-L1 cells were treated with 10 µM PP1 for 30 min before stimulation with 10 nM IGF-I for 1 min (Pyk2) or 5 min (MAPK). Pyk2 was immunoprecipitated from cell lysates and analyzed by Western blot using antiphosphotyrosine AG10 antibodies (PY) followed by anti-Pyk2 antibodies (Pyk2). Cell lysates were analyzed by Western blot for active MAPK [phospho-MAPK (P-MAPK)] and ERK-1/2 (total MAPK) in the presence or absence of PP1 to demonstrate the efficacy of PP1.
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Pyk2 Has a Role in IGF-I Activation of Src and MAPK
To determine the importance of Pyk2 in IGF-I signaling to Src and the downstream MAPK pathway, we treated cells with the intracellular calcium chelator BAPTA, which has been shown to inhibit Ca-dependent Pyk2 activation in platelets (40), vascular smooth muscle cells (41), cardiac fibroblasts (42), and osteoclasts (43). Cells were pretreated for 30 min with 50 µM BAPTA and then stimulated with 10 nM IGF-I. Cells treated with IGF-I for 1 min were used for immunoprecipitation of Pyk2 and Src, and cells treated with IGF-I for 5 min were used for analysis of MAPK activation. Figure 6
shows the Western blots for Pyk2 PY-402, Src PY-418 and phosphorylated MAPK. Blots were stripped, and Western analysis for total Pyk2, Src and MAPK indicated no differences in protein content between BAPTA or IGF-I-treated and untreated cells. We found that BAPTA inhibited IGF-I-stimulated Pyk2 autophosphorylation, indicating IGF-I activation of Pyk2 is calcium dependent. BAPTA also prevented IGF-I-stimulated phosphorylation of Src tyrosine 418 and MAPK but had no effect on basal MAPK, suggesting that IGF-I-activated Src and MAPK are also dependent on intracellular calcium possibly through Pyk2 activation.

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Fig. 6. Calcium Chelation Inhibits IGF-I Phosphorylation of Pyk2, Src, and MAPK
Serum-starved subconfluent 3T3-L1 cells were treated with 50 µM BAPTA for 30 min before stimulation with 10 nM IGF-I for 1 min for Pyk2 and Src analysis and 5 min for MAPK. Pyk2 and Src were immunoprecipitated from cell lysates and analyzed by Western blot using anti-PY402-Pyk2 antibodies or anti-PY418-Src antibodies, followed by anti-Pyk2 or anti-Src antibodies. Cell lysates were analyzed by Western blot using phospho-MAPK antibodies (P-MAPK) followed by ERK-1/2 antibodies.
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Calcium chelation is a nonspecific mechanism of Pyk2 inhibition; therefore, to more directly inhibit Pyk2 activity, we transiently expressed wild-type Pyk2 (CADTK) or a dominant-negative form of Pyk2 (CRNK) into subconfluent 3T3-L1 cells. CRNK contains only the C-terminal portion with the focal adhesion targeting domain and lacks the tyrosine-402 autophosphorylation site, which is also the Src binding site (37). This mutant has been shown to abolish Pyk2 function in 3T3-L1 adipocytes (16). Forty-eight hours after transfection, cells were treated with 10 nM IGF-I for 5 min and MAPK activity determined by Western blot (Fig. 7
). CADTK had no effect on IGF-I activation of MAPK. However, CRNK inhibited IGF-I activation of MAPK. The degree of MAPK inhibition correlated with the extent of transfection, i.e. approximately 50% inhibition under conditions that achieved a transfection efficiency of approximately 50%.

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Fig. 7. Inhibition of Pyk2 Impairs IGF-I Activation of MAPK
Serum-starved subconfluent 3T3-L1 cells were transiently transfected with no Pyk2 DNA (mock), CADTK or CRNK and allowed to recover for 24 h. All cells were cotransfected with green fluorescent protein as a marker of transfection: if more than 50% of cells expressed green fluorescent protein by fluorescence microscopy at 2436 h, then cells were serum starved overnight before stimulation with 10 nM IGF-I for 5 min. Cell lysates were analyzed by Western blot for phospho-MAPK (P-MAPK) and ERK-1/2 MAPK (total MAPK). This experiment was repeated twice with comparable results.
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DISCUSSION
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We have previously shown that membrane-associated Src is required for IGF-I activation of the ERK-1 and -2 MAPK pathway in 3T3-L1 cell proliferation (4, 5). The primary goal of the present study was to determine the mechanism of Src activation that links IGF-I to MAPK in proliferating 3T3-L1 cells. There is now abundant evidence that IGF-I signaling pathways mediating cell growth involve interaction of the IGF-I receptor (IGFR) with membrane-associated multiprotein complexes, including integrins, integral membrane proteins and focal adhesion proteins (18, 19, 44). We have shown that
vß3 integrins are necessary for IGF-I activation of Src and MAPK, which is consistent with data on IGF-I signaling in smooth muscle cells (23, 25, 45). However, we have also demonstrated that IGF-I increases integrin phosphorylation and ligand binding, directly supporting the model of inside-out activation of
vß3 integrins. Indirect activation of integrins by growth factor receptors is thought to occur through intracellular signals promoting increased extracellular ligand binding. This phenomenon of inside-out activation of integrins is well described (21, 46) and consistent with IGF-I action in smooth muscle (23) and trophoblasts (28) and now 3T3-L1 cells, suggesting that IGF-I inside-out activation of integrins may be a general mechanism of IGF-I signaling.
We have not investigated the molecular interaction between the activated IGFR and
vß3 integrins. Studies of IGF-I signaling mediated by ß1 integrins have demonstrated complex formation between the IGFR and ß1 subunits and dependence of full IGFR phosphorylation on integrin activation (47, 48). In contrast, a direct association between the IGFR and
vß3 integrins has not been found (49), although
vß3 integrin activation was also necessary for normal IGFR phosphorylation in smooth muscle cells and osteoprogenitor cells (50). In smooth muscle cells, the mechanism of
vß3 integrin activation of the IGFR involves integral membrane proteins and regulation of the phosphatase SHP-2, which dephosphorylates the IGFR (19). We have not pursued studies related to potential roles of integrin-associated proteins involved in the action of
vß3 integrins on IGFR phosphorylation or direct interaction of
vß3 integrins and the IGFR in 3T3-L1 cells, because one limitation has been the lack of anti-ß3 subunit antibodies suitable for immunoprecipitation of mouse ß3 subunit. However, our results considered together with data from Clemmons et al. (19) suggest a reciprocal, not one-way, relationship between the IGFR and
vß3 integrins.
Given our observation that
vß3 integrins are required for IGF-I activation of Src and MAPK, we investigated potential integrin-associated focal adhesion signaling components involved in activating Src and MAPK. FAK has been shown to be a substrate for both the insulin receptor and IGFR (51), and IGF-I-activated FAK has been shown to mediate IGF-I activation of MAPK in some cells (52) and not others (53). We found a small but reproducible increase above basal in FAK tyrosine phosphorylation with IGF-I, but no appreciable increased association of FAK and Src. However, coimmunoprecipitation is a crude and not particularly sensitive method of detecting direct interaction between proteins. Our data suggest that FAK may have a role in mediating IGF-I activation of Src and MAPK, but further investigation needs to be performed. However, our data could be interpreted to suggest that integrin-associated FAK and Src mediate basal MAPK activity in attached 3T3-L1 cells and that the small increase in FAK phosphorylation with IGF-I stimulation is secondary to IGF-I-activated Src and/or Pyk2 because both Src (54, 55) and Pyk2 (37, 56) have been shown to phosphorylate FAK.
The related FAK Pyk2, expressed in hematopoietic and neuronal cells (14) as well as 3T3-L1 adipocytes (16), is activated by a variety of extracellular signals to form a complex with Src and regulate downstream signaling pathways, including MAPK. Upon activation, Pyk2 trans-autophosphorylates tyrosine 402, which is the Src binding site, resulting in recruitment and activation of Src (35). We have demonstrated that activated Pyk2 associates with Src in an IGF-I-dependent manner and that specific inhibition of Pyk2 inhibits IGF-I activation of MAPK. These data indicate that IGF-I signaling to MAPK is dependent on integrin-facilitated multiprotein complex formation involving Pyk2 and Src. The importance of Pyk2/Src complex formation in
vß3 integrins signaling has been extensively studied in osteoclasts (38, 43, 57). In addition, cross talk between integrins and G protein-coupled receptors and growth factor tyrosine kinase receptors has been shown to involve Pyk2/Src complexes (17, 42). Although our data are the first to demonstrate a role for Pyk2/Src complexes in IGF-I mitogenic signaling, Pyk2 has been previously shown to be required for IGF-I-stimulated neurite formation, although those investigators did not study the signaling complexes or pathways mediating neurite growth (44).
As in many other cell types, we have shown that IGF-I activation of Pyk2 is calcium dependent, although the mechanism of Pyk2 activation in response to IGF-I remains unclear. Based on our data demonstrating a requirement for
vß3 integrin activation, one might hypothesize IGF-I-stimulated integrin-dependent recruitment of Pyk2 to focal adhesion complexes. The translocation of Pyk2 to focal adhesions has been shown to be mediated by its carboxyl-terminal domain, also called the focal adhesion targeting domain (34). Pyk2 has been found to require and directly interact with the ß3 subunit in some cells (58) but not in others (59). Our studies indicate that activation of Pyk2 and Src is calcium dependent, suggesting a potential role for protein kinase C (PKC) in IGF-I activation of Pyk2 and subsequent complex formation with Src. PKC mediates Pyk2 activation and focal adhesion targeting in fibronectin-induced integrin signaling in chondrocytes (60) as well as G protein-coupled receptor-mediated activation in other cells (15, 34). It remains to be determined whether PKC plays a role in IGF-I-activated Pyk2 in 3T3-L1 cells and whether
vß3 integrins directly or indirectly regulate Pyk2 activation and Src association by IGF-I.
We have previously shown that membrane-associated Src mediates IGF-I activation of MAPK in proliferating 3T3-L1 cells. Based on the current study, IGF-I signaling to the MAPK pathway is dependent on
vß3 integrin activation and formation of a signaling complex comprised of the calcium-dependent FAK Pyk2 and Src. Our results strongly support an emerging model of the IGFR acting in concert with integrin-associated proteins such as Src, FAK, and Pyk2 to control cell growth. Considering the results from Clemmons et al. (19) that
vß3 integrins regulate IGFR function and downstream signaling and our results that indicate IGF-I regulates
vß3 activation and association of Pyk2 and Src, reciprocal cross talk involving multiprotein complexes with the IGFR and integrins may be a common mechanism of IGF-I-mediated cell growth.
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MATERIALS AND METHODS
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Materials
Tissue culture reagents were purchased from Invitrogen Life Technologies (Grand Island, NY). Buffer reagents, echistatin, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA/AM), Kodak X-Omat AR film, and anti-FLAG antibody were purchased from Sigma (St. Louis, MO). Enhanced chemiluminescence reagents, Hyperfilm ECL, and Hybond C nitrocellulose were purchased from Amersham Life Science (Arlington Heights, IL). Polyvinylidene difluoride membranes were purchased from Bio-Rad Laboratories (Hercules, CA). Src antibodies were purchased from Oncogene Research Products (Cambridge, MA). Antibodies to MAPK-1/2 (ERK1/2-CT), phosphotyrosine clone 4G10, FAK, Pyk2, and myc tag were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies to dual phosphorylated ERK-1 and -2 MAPK and phospho-Pyk2 [pY402] were purchased from Cell Signaling Technology (Beverly, MA). Integrin ß3 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and antiintegrin ß1 and antiintegrin
v were from Chemicon (Temecula, CA). Anti-FAK[pY397], antiintegrin ß3[pY773] and anti-Src[pY418] were from BioSource International (Camarillo, CA). LIBS antibodies were a generous gift from Dr. Mark Ginsberg (Scripps Research Institute, La Jolla, CA). Human recombinant IGF-I was obtained from GroPep (Adelaide, Australia). PP1 was purchased from BioMol (Plymouth Meeting, PA).
Cell Culture
The murine preadipocyte line 3T3-L1 was obtained from American Type Culture Collection (Manassas, VA) and cultured as previously described (4). Cell monolayers were cultured in DMEM with 10% fetal bovine serum in 10-cm dishes for immunoprecipitation studies and six-well plates for Western blotting of cell lysate proteins. Monolayers were used at 5070% confluence and placed in serum-free DMEM with 0.1% BSA overnight before IGF-I treatment and analysis.
Immunoprecipitation and Western Blot Analysis
Total cellular lysates were obtained using RIPA buffer with 1% Nonidet P-40 and 0.1% sodium dodecyl sulfate for tyrosine protein kinase analyses as described previously (4) or 0.2% Triton X-100 lysis buffer for MAPK analysis as described previously (2). Cell lysates containing 350500 µg total protein were used for immunoprecipitation. For Western blot analysis of MAPK, 20 µg total cell lysate protein from 50% confluent cells was used. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose for MAPK or polyvinylidene difluoride membranes for tyrosine kinases, blocked in 5% BSA in Tris-buffered saline with 0.1% Triton X-100 and probed with primary antibody at 1 µg/ml. Specific binding was visualized using enhanced chemiluminescence and Hyperfilm ECL and then analyzed by digital image analysis using a Hewlett-Packard ScanJet 6100C/T scanner with Gel Pro Analyzer 3.1 software from Media Cybernetics (Silver Spring, MD).
LIBS Immunocytochemistry
Cell monolayers were grown in two-well chamber slides, fixed in 100% methanol for 10 min at room temperature (RT), blocked in 10% normal goat serum for 20 min at RT, and then incubated with 1:100 dilution of anti-LIBS in 10% goat serum for 1 h at RT. The monolayers were washed with PBS and incubated for 45 min at RT in the secondary antibody goat antimouse Alexa-Fluor (Molecular Probes, The Netherlands). After additional washing, coverslips were mounted with Vectashield plus 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and random fields of view visualized as described previously (3). Controls included omission of the primary antibody and normal mouse IgG instead of the primary antibody.
Transient Transfection
Cells were cultured in six-well plates and used at approximately 50% confluence. The pcDNA3 plasmids, kindly provided by Dr. H. S. Earp (University of North Carolina at Chapel Hill), included myc-tagged CADTK or a dominant-negative FLAG-tagged form of Pyk2 containing the carboxy terminus of the protein (CRNK) (37). CADTK or CRNK was transfected at 1 µg/well using Gene Porter 2 (Gene Therapy Systems, San Diego, CA) in 10% serum-containing medium overnight at 37 C. Cotransfection of the plasmid pEGFP-F expressing a farnesylated green fluorescent protein (CLONTECH, Palo Alto, CA) was used at 0.1 µg/well as a marker of transfection efficiency that ranged from 4060%. The negative control was a mock transfection, i.e. the transfection protocol was performed but no Pyk2 DNA, only pEGFP-F was added. The day after transfection, medium is changed to standard 10% serum-containing medium. Cells are used for analyses 24 h later, when they are still less than 80% confluent.
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ACKNOWLEDGMENTS
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We thank Dr. Jennifer Farrington and Dr. Molly Harrington for technical assistance, and Dr. Robert J. Smith and Dr. Philip Gruppuso for advice and review of the manuscript.
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FOOTNOTES
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This work was supported by National Institutes of Health Grant RO1 DK59339 and Rhode Island Hospital Department of Pediatrics Research Endowment.
First Published Online March 10, 2005
Abbreviations: BAPTA, Bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid; CADTK, wild-type Pyk2; CRNK, dominant-negative form of Pyk2; FAK, focal adhesion kinase; IGFR, IGF-I receptor; LIBS, ligand-induced binding site; PKC, protein kinase C; Pyk-2, calcium-dependent proline-rich tyrosine kinase-2; RT, room temperature.
Received for publication November 30, 2004.
Accepted for publication March 1, 2005.
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