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Cell Growth & Differentiation Vol. 12, 379-386, July 2001
© 2001 American Association for Cancer Research

Src Family Tyrosine Kinases Participate in Insulin-like Growth Factor I Mitogenic Signaling in 3T3-L1 Cells1

Charlotte M. Boney2, Hiroko Sekimoto, Philip A. Gruppuso and A. Raymond Frackelton, Jr.

Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, Rhode Island 02903 [C. M. B., H. S., P. A. G.]; Department of Medicine, Roger Williams Hospital, Providence, Rhode Island 02908 [A. R. F.]; and Department of Pathology and Laboratory Medicine, Brown University [A. R. F.], Providence, Rhode Island 02903


    Abstract
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Insulin-like growth factor-I (IGF-I) stimulates proliferation and differentiation of many cell types, including preadipocytes. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of the extracellular regulated kinase (ERK)-1 and -2 mitogen-activated protein kinase (MAPK) pathway, and that IGF-I-stimulated MAPK is predominantly downstream of Shc, not IRS-1 phosphorylation. The Src family of nonreceptor tyrosine kinases has been shown to mediate the mitogenic effects of other growth factors that also activate Shc and the ERK-1 and -2 MAPKs. Although Src family kinases (SFK) have been implicated in IGF-I action, no specific role for SFKs in IGF-I regulation of mitogenesis has been previously demonstrated. We studied the role of SFKs in IGF-I mitogenic signaling in 3T3-L1 preadipocytes. The SFK-selective inhibitor PP1 completely inhibited both IGF-I-stimulated DNA synthesis and MAPK activation in proliferating 3T3-L1 cells. PP1 inhibited IGF-I phosphorylation of Shc but not of IRS-1. In addition, IGF-I activation of MAPK was inhibited in proliferating cells transiently transfected with a dominant-negative c-Src. Finally, the kinetics of SFK and MAPK activation by IGF-I suggest that SFKs may act upstream of MAPK. IGF-I activation of SFK members c-Src and Fyn occurred within 1 min of treatment, and activity was back to baseline by 10 min. Our previous studies found that IGF-I activation of MAPK peaked at 5 min and was also back to baseline by 10 min. Our results are the first to demonstrate that SFKs mediate IGF-I mitogenic signaling in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.


    Introduction
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
IGF-I3 plays an important role in cell growth both in vitro and in vivo (1 , 2) . IGF-I stimulates mitogenesis in a number of cell types in culture, including fibroblasts (3) and preadipocytes (4) . The biological actions of IGF-I are mediated by its receptor tyrosine kinase, the IGFR (5) . Binding of IGF-I to the IGFR leads to receptor autophosphorylation followed by tyrosine phosphorylation of substrates such as the IRS proteins and Shc (5) . These adaptor proteins can bind Grb2/mSOS to activate the Ras-MAPK pathway (6) . IRS-1 can bind multiple adaptor proteins for activation of numerous downstream pathways, such as PI3K (7) .

Increasing evidence suggests that IRS-1 and Shc mediate distinct roles in IGF-I action. We have previously shown that IGF-I-stimulated mitogenesis in 3T3-L1 preadipocytes involves Shc phosphorylation that, in turn, mediates MAPK activation (8) . Although a role for IRS-1 in IGF-I mitogenic signaling has been identified in some cells (9, 10, 11) , substantial evidence exists for Shc mediating most of the mitogenic effects of IGF-I (12, 13, 14) .

We have previously observed that IGF-I stimulation of Shc and MAPK, but not IRS-1, is lost when density-induced, growth-arrested 3T3-L1 preadipocytes are stimulated to differentiate (15) . This loss of IGF-I mitogenic signaling suggests modulation in the pathway from the IGFR to MAPK. Modulation of IGF-I mitogenic signaling has been demonstrated to occur at the receptor or downstream of the receptor. IGFR internalization regulates receptor tyrosine kinase activity (16) . Regulation of IGFR signaling pathways occurs via transactivation of the EGF receptor (17) or activation of protein kinases such as Src tyrosine kinases (18) .

Although the transforming gene v-src has been extensively studied in cancer, it is now well established that the normal gene, c-src, is part of a family of Src kinases that plays an important role in normal cell growth. SFKs mediate mitogenesis by several growth factors that activate the Shc-MAPK signaling pathway, including EGF and PDGF (19 , 20) . No role for SFKs in IGF-I-mediated growth has been demonstrated. However, SFKs have been implicated in IGF-I action. The IGFR is a substrate for Src in vitro (18) , Src increases IGFR number (21) , and a relationship between IGF-I and v-src is well established in neoplastic transformation (22) . A role for SFKs in IGF-I signaling has been shown in fibroblasts (23) and in regulation of neuronal calcium channels (24) .

Because of its role in growth factor-mediated mitogenic signaling in many cell types (19) , we hypothesized that SFKs participate in IGF-I mitogenic signaling in 3T3-L1 cells. Using selective inhibitors of the Src kinases, we show in this report that SFKs are involved in IGF-I-stimulated DNA synthesis, Shc phosphorylation, and MAPK activation in 3T3-L1 cells. We show that IGF-I directly stimulates SFK activity. To our knowledge, this is the first demonstration that IGF-I regulates mitogenesis through a signaling pathway involving SFKs.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
The SFK Inhibitor PP1 Inhibits IGF-I-stimulated DNA Synthesis.
We have previously shown that inhibition of IGF-I-stimulated Shc phosphorylation or MAPK activation inhibits DNA synthesis in proliferating 3T3-L1 cells, suggesting that the Shc-MAPK pathway mediates IGF-I mitogenesis in 3T3-L1 cells (8) . To determine whether the SFKs play a role in IGF-I mitogenesis, we analyzed DNA synthesis after treatment with the cell-permeable, tyrosine kinase inhibitor PP1 (25) . This Src family-selective tyrosine kinase inhibitor binds to the threonine residue at position 338 common to all cellular SFK members, preventing binding of ATP (26) . We treated subconfluent, serum-starved 3T3-L1 cells with 10 nM IGF-I in the presence or absence of 10 µM PP1 for 24 h and then measured DNA synthesis by [3H]thymidine incorporation (Fig. 1)Citation . PP1 inhibited IGF-I-stimulated [3H]thymidine incorporation by ~90%, suggesting that SFKs play a role in IGF-I-mediated 3T3-L1 mitogenesis. However, PP1 also inhibited basal [3H]thymidine incorporation, consistent with an IGF-I-independent role of SFKs. It is well established that SFKs are involved in multiple cellular functions involved in growth that do not require IGF-I, such as in mitosis (27) .



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Fig. 1. Src kinase inhibitor PP1 inhibits [3H]thymidine incorporation by IGF-I. Proliferating 3T3-L1 cells were treated with or without 10 µM PP1 in the presence of 10 nM IGF-I (solid bars) or no hormone (open bars) for 24 h. [3H]thymidine (1 µCi/well) was added for the last 4 h. Cells were lysed in 0.33 M NaOH, an aliquot was removed for protein concentration determination, and DNA was precipitated with cold TCA. n = 6 wells (samples) per condition. This experiment was repeated once with identical results.

 
The SFK Inhibitor PP1 Inhibits IGF-I-stimulated Mitogenic Signaling.
SFKs have been shown to mediate DNA synthesis in fibroblasts by several growth factors, including EGF and PDGF (20 , 28) . SFK members are activated by these receptor tyrosine kinases, and Shc may be the key substrate that links SFKs to the MAPK pathway in growth factor signaling (29) . This is consistent with our earlier findings that IGF-I mitogenic signaling in 3T3-L1 cells involves the Shc-MAPK pathway (8) . In addition, except for yeast two-hybrid studies indicating a direct association of Shc with the IGFR (30) , there is little evidence that Shc as a direct substrate of the activated IGFR.

To determine whether the effects of PP1 on IGF-I-stimulated mitogenesis are associated with inhibition of IGF-I mitogenic signaling, we first analyzed ERK-1 and -2 MAPK activation by IGF-I after PP1 treatment. Proliferating 3T3-L1 cells were treated with 10 µM PP1 for 30 min and then were stimulated with 10 nM IGF-I for 5 min prior to analysis by Western blotting for phospho-specific ERK-1 and -2. Similar to its effect on DNA synthesis, PP1 completely inhibited basal MAPK activity, and IGF-I-stimulated MAPK activity was inhibited to baseline levels in 3T3-L1 cells (Fig. 2)Citation . This is consistent with our previous observation that inhibition of IGF-I-stimulated MAPK activation by PD098059, a selective inhibitor of the MAPK kinase, MEK, inhibits DNA synthesis by 80–90% (8) . These data support the conclusion that IGF-I-stimulated mitogenesis is via the MAPK pathway, and that MAPK activation by IGF-I involves SFKs.



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Fig. 2. Src kinase inhibitor PP1 inhibits MAPK activation by IGF-I. Proliferating 3T3-L1 cells were treated with 10 µM PP1 for 30 min prior to stimulation with 10 nM IGF-I (solid bars) for 5 min or no stimulation (open bars). Proteins in cell lysates were separated by SDS-PAGE and analyzed by Western blotting using phospho-MAPK antibodies (top panel) followed by ERK1/2 MAPK antibodies (not shown). Below the blot, densitometry of the phospho-MAPK bands, corrected for total MAPK content. This experiment was repeated two more times with the same results.

 
We have previously shown that ERK-1 and -2 MAPK activation by IGF-I in proliferating 3T3-L1 cells is predominantly via phosphorylation of Shc and not of IRS-1 (8) . To determine the effects of PP1 on these upstream activators of MAPK, we analyzed tyrosine phosphorylation of Shc and IRS-1 in proliferating and postconfluent growth-arrested 3T3-L1 cells. After treatment with 10 µM PP1 for 30 min, cells were stimulated with 10 nM IGF-I, and cell lysates were generated for immunoprecipitation and Western blotting of tyrosine phosphorylated Shc and IRS-1. Consistent with our previous observations (15) , IGF-I-stimulated Shc phosphorylation is decreased in growth-arrested 3T3-L1 cells (Fig. 3)Citation . PP1 completely abolished Shc phosphorylation by IGF-I in proliferating cells (Fig. 3)Citation . Although PP1 also inhibited Shc phosphorylation by IGF-I in growth arrested cells, the difference was not statistically significant. However, we have previously shown that IGF-I activation of Shc in growth-arrested 3T3-L1 cells is significantly decreased (15) ; therefore, we would expect the magnitude of the difference in the PP1-treated and untreated groups to be small. Finally, PP1 had no effect on IGF-I-stimulated IRS-1 tyrosine phosphorylation or coimmunoprecipitated p85 subunit of PI3K (Fig. 3)Citation . The differential effects by PP1 on these two substrates of the IGF-I receptor suggest that the Shc/MAPK and IRS-1/PI3K pathways are distinct cascades, and that Shc, but not IRS-1, is a substrate of the SFKs.



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Fig. 3. Src kinase inhibitor PP1 inhibits Shc but not IRS-1 tyrosine phosphorylation by IGF-I. A, proliferating and growth-arrested 3T3-L1 cells were treated with or without 10 µM PP1 for 30 min prior to stimulation with 10 nM IGF-I for 5 min. The immunoprecipitates (IP) were resolved by SDS-PAGE and transferred to membranes. Top two panels, Western blots of Shc IPs exposed first to phosphotyrosine antibodies, then stripped and exposed to Shc antibodies. Bottom three panels, Western blots of IRS-1 IPs exposed first to phosphotyrosine, then p85, and finally IRS-1 antibodies. B, IGF-I-stimulated phosphotyrosine (PY) of Shc and IRS-1, compiled from three separate experiments and corrected for total p52 Shc and total IRS-1, was analyzed by densitometry of Western blots and expressed as fold increase (mean + SE) in IGF-I-stimulated PY from control cells (open bars) or PP1-treated cells (solid bars). The differences in the PP1-treated and untreated groups were analyzed using ANOVA with a Tukey-Kramer post hoc test. *, significance at P < 0.001.

 
These data suggest that SFKs play a role in IGF-I-stimulated phosphorylation of Shc and support our earlier conclusion that MAPK activation by IGF-I is predominantly downstream of Shc, not of IRS-1, in proliferating 3T3-L1 cells. These results are consistent with other studies demonstrating Shc phosphorylation and activation of the Ras-MAPK pathway by growth factor-activated SFKs (19) . However, our conclusions are based on the assumption that inhibitory effects of low concentrations of PP1 are highly selective for SFKs compared with other tyrosine kinases or serine/threonine kinases (25 , 26) . The absence of PP1 inhibition of IGF-I-activated IRS-1 indicates that IGF-I receptor tyrosine kinase activity is unaffected (Fig. 3)Citation . PP1 has been shown to inhibit the PDGF receptor tyrosine kinase (31) , but when we treated 3T3-L1 cells with the selective PDGF receptor inhibitor tyrphostin AG-1296, we saw no inhibition of IGF-I-stimulated MAPK activity but we did see appropriate inhibition of PDGF-BB-stimulated MAPK activity (data not shown).

Although PP1 appears to have no effect on IRS-1 activation, in that changes in tyrosine phosphorylation are not detectable and signaling to PI3K appears to be intact, Western blotting may not detect small changes in the 30 potential tyrosine phosphorylation sites of the IRS-1 protein. In addition, IRS-1 can bind multiple signaling molecules at once; therefore, detecting p85 subunit association does not indicate its binding of other molecules, such as Grb2 (7) . It is possible that the degree of IGF-I-stimulated MAPK activation not inhibited by PP1 is IRS-1 dependent and is mediated through binding of Grb2. Given the observation by Sun et al. (32) that IRS-1 associates with the SFK member Fyn in Chinese hamster ovary cells stimulated with insulin, it is possible that SFKs are involved in IGF-I-activated IRS-1 signaling in proliferating preadipocytes.

A Doubly Mutated, Dominant-Negative Src Inhibits IGF-I-stimulated Mitogenic Signaling.
To confirm that the inhibitory effects on IGF-I signaling by PP1 are specific for the SFKs, we transiently transfected proliferating 3T3-L1 cells with wild-type c-Src or a doubly mutated, catalytically inactive c-Src before analysis of IGF-I mitogenic signaling. This dominant-negative Src mutant has mutations of lysine-295 to arginine and tyrosine-527 to phenylalanine (SrcRF), which inactivate the ATP-binding site and disrupt intramolecular folding (33) . Transfection efficiency was ~70% as determined by cotransfection of green fluorescent protein. MAPK activation was determined 24 h after transfection. Fig. 4ACitation shows a representative Western blot of MAPK activation by IGF-I in control cells and cells transfected with wild-type c-Src and the Src mutant, SrcRF. Fig. 4BCitation shows densitometry results of phospho-MAPK, corrected for total MAPK content, from two experiments including the one shown in Fig. 4ACitation . In wild-type c-Src transfected cells, baseline MAPK activity is increased, and IGF-I stimulation of MAPK is by ~3-fold. However, in cells transfected with the kinase-dead SrcRF mutant, IGF-I-stimulated MAPK activity is almost completely inhibited. These results are consistent with the results of the PP1 experiments and provide more conclusive evidence that SFKs mediate IGF-I mitogenic signaling in proliferating 3T3-L1 cells.



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Fig. 4. Dominant-negative SrcRF mutant inhibits IGF-I-stimulated MAPK activation. A, proliferating 3T3-L1 cells were mock-transfected (control) or transfected with wild-type c-Src (WT c-src) or SrcRF mutant (srcRF) at a transfection efficiency of ~70%. Twenty-four h after transfection, cells were incubated overnight in SFM and then stimulated with 10 nM IGF-I for 5 min. Cell lysates were analyzed for MAPK activation by Western blotting for phospho-MAPK (A) and then total MAPK (not shown). B, phospho-MAPK unstimulated (open bars) and after IGF-I stimulation (solid bars) from two separate transfection experiments (n = 4, each condition) was normalized for the total amount of MAPK and expressed as densitometric units (mean + SE).

 
IGF-I Stimulates SFK Activity in 3T3-L1 Cells.
Inhibition of IGF-I-stimulated MAPK activation by PP1 and the SrcRF mutant clearly implicate SFK involvement in IGF-I mitogenic signaling. If SFKs participate in IGF-I mitogenic signaling and are directly downstream of IGFR activation, then IGF-I should stimulate the activity of one or more SFK members. Direct activation of c-Src by IGF-I has been shown in neuroblastoma cells (24) , and interestingly, Arbet-Engels et al. demonstrated inhibition of c-Src activity by IGF-I in NIH 3T3 cells (23) . We sought to identify the SFK member activated by IGF-I in 3T3-L1 cells that might be participating in IGF-I mitogenic signaling. Given that the three ubiquitously expressed SFK members c-Src, Fyn, and Yes have been previously identified in 3T3-L1 cells (34) , we immunoprecipitated Src, Fyn, and Yes from IGF-I-stimulated proliferating cells and measured kinase activity with an in vitro assay using enolase as a substrate (35) . IGF-I stimulated c-Src kinase activity in proliferating cells at a peak stimulation time of 1 min (Fig. 5A)Citation . The kinetics of IGF-I-stimulated c-Src and MAPK are consistent with SFK activation occurring upstream of MAPK: both activities are back to baseline by 10 min, and our previous studies demonstrated peak MAPK activation was at 5 min (15) . IGF-I stimulation of c-Src and Fyn activity was comparable, ~3-fold more than baseline, and dependent on immunoprecipitation with specific antibodies (Fig. 5B)Citation . IGF-I activation of both c-Src and Fyn is not surprising, given the redundancy among the SFKs that has been demonstrated in various SFK knockout models (36) .



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Fig. 5. IGF-I stimulates c-Src and Fyn kinase activities. A, proliferating 3T3-L1 cells were serum starved overnight, then stimulated with 10 nM IGF-I. Top panel, a representative autoradiogram of enolase phosphorylation by immunoprecipitated c-Src from cells stimulated with IGF-I for 0, 1, 2, 5, and 10 min. The graph is a compilation of densitometry results (mean + SD) of three time course experiments; results were corrected for a zero time of 1 densitometry unit. B, kinase assay autoradiograms with corresponding Western blots of c-Src or Fyn from cells stimulated with 10 nM IGF-I for 1 min. An aliquot of immunoprecipitated c-Src or Fyn was removed for Western blotting prior to measurement of kinase activity using enolase (arrow) as substrate. Immunoreactive c-Src and Fyn are seen just above immunoglobulin heavy chain at Mr = 60,000 and 58,000, respectively (arrows). Controls, which used IGF-I-stimulated cell lysates, included immunoprecipitation with nonimmune IgG (Control i.p.), No sample (omission of immunoprecipitate), and no enolase (No substrate).

 
Multiple attempts to immunoprecipitate Yes using three different antibodies resulted in barely detectable Yes protein by Western blot and no detectable activity by the kinase assay (data not shown), suggesting low levels of Yes protein expression in 3T3-L1 cells. All three of the antibody preparations used for immunoprecipitation were raised against a Yes peptide of human sequence, and the mouse and human sequences differ by only a single amino acid (37) .

Given the evidence that SFKs mediate EGF mitogenic signaling in fibroblasts (20 , 28) and the recent report of IGF-I-activated Shc and MAPK via transactivation of the EGF receptor in COS-7 cells (17) , we tested the hypothesis that IGF-I stimulates the MAPK pathway via transactivation of the EGF receptor. We treated proliferating 3T3-L1 cells with the selective EGF receptor kinase inhibitor tyrphostin AG-1478 at 50 µM for 2 h, and then compared MAPK activity by Western blot after stimulation with 10 nM IGF-I or EGF for 5 min. We found no inhibition of IGF-I-stimulated MAPK activation, but EGF-stimulated MAPK activity was inhibited as expected (data not shown). Although cross-talk between the EGF receptor and the IGFR has been demonstrated in several cell types (17 , 38) , our data indicate that IGF-I activation of MAPK in 3T3-L1 cells is not via transactivation of the EGF receptor.

Heterotrimeric inhibitory G proteins have been shown to modulate IGF-I mitogenic signaling in rat1 fibroblasts (39) and neuronal cells (40) . Insulin-stimulated MAPK activity may be partially PT sensitive in 3T3-L1 cells (41) . These data suggest a role for Gi proteins in IGF-I mitogenic signaling; therefore, we analyzed MAPK activation from proliferating 3T3-L1 cells treated overnight with 100 ng/ml or 200 ng/ml PT prior to stimulation with 10 nM IGF-I for 5 min. We saw no effect of PT at 100 ng/ml (data not shown), but at 200 ng/ml, baseline MAPK activity and IGF-I-stimulated MAPK activation were inhibited to a similar degree (Fig. 6)Citation . The densitometric analysis of the Western blots indicates that IGF-I stimulates MAPK activity about 10-fold in untreated cells and about 13-fold in PT-treated cells. We interpret these results to suggest that PT-sensitive MAPK activity is independent of IGF-I; therefore, Gi proteins do not appear to play a significant role in IGF-I-dependent activation of MAPK.



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Fig. 6. IGF-I-stimulated MAPK activation in the presence of PT. Proliferating 3T3-L1 cells were treated with 200 ng/ml PT for 16 h prior to stimulation with (solid bars) or without (open bars) 10 nM IGF-I for 5 min. Proteins in cell lysates were separated by SDS-PAGE and analyzed by Western blotting using phospho-MAPK antibodies (P-MAPK) followed by ERK1/2 MAPK antibodies (Total MAPK). The corresponding densitometry of the blots is expressed as mean + SE of phospho-MAPK corrected for total MAPK.

 
The stimulatory G protein subunit Gs{alpha} has recently been shown to directly activate Src by changing its conformation (42) . Although our data suggest that Gi proteins do not have a major role in IGF-I mitogenic signaling in 3T3-L1 cells, others have shown that the inhibition of Gs{alpha} in 3T3-L1 cells accelerates differentiation (43) . Given our prior observation that the inhibition of MAPK activation accelerates differentiation (8) , these observations led us to speculate that Gs{alpha} may have a role in SFK-mediated IGF-I mitogenic signaling. We measured MAPK activity in proliferating 3T3-L1 cells after overnight treatment with 10 mg/ml cholera toxin, an activator of Gs{alpha}, but we found no stimulation of MAPK (data not shown). These results suggest that Gs{alpha} modulation of differentiation is independent of MAPK activation, and that Gs{alpha} is probably not mediating IGF-I-activated SFKs and downstream mitogenic signaling.

It is now well established that SFKs play an important role in cell proliferation and differentiation in many cell types as well as in vivo. Animals deficient in various SFK members have a restricted phenotype, suggesting compensatory actions among the kinases (36) . However, animals deficient in multiple SFK members, such as src/fyn or src/yes double mutants, die perinatally (44) .

Receptor tyrosine kinase mitogenic signaling through SFKs has been established for a variety of growth factors, including EGF, PDGF, colony-stimulating factor, and fibroblast growth factor (19) . SFK involvement in IGF-I action has been demonstrated in fibroblasts (23) , v-Src transformation of IGFR-positive mouse embryo fibroblasts (18 , 22) , and IGF-I-potentiated calcium channel currents in neurons (24) . Our data indicating activation of c-Src and Fyn by IGF-I in 3T3-L1 preadipocytes is similar to the 2- to 3-fold activation of c-Src by IGF-I in neuroblastoma cells (24) and by EGF in several other cell types (45) .

Despite the abundant data establishing the role of SFKs in growth factor-initiated mitogenesis, the mechanism for SFK activation is unclear. SFKs can be activated by tyrosine phosphorylation of specific sites or by conformational change (19 , 46) . There is evidence of direct phosphorylation of SFKs by the activated EGF and PDGF receptors in fibroblasts (19) and evidence of indirect activation of SFKs mediated through integrins (47) , G-protein coupled receptors (48) , and ß-arrestins (49, 50, 51) , probably through formation of large signaling complexes. In the present study, the mechanism of IGF-I-activated SFKs and downstream mitogenic signaling is not clear but appears not to involve G proteins. These data demonstrate for the first time that SFKs mediate IGF-I mitogenesis in 3T3-L1 cells and add to the growing body of evidence that SFKs play a crucial role in IGF-I action.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Materials.
Tissue culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Buffer reagents, enolase, and Kodak X-Omat AR film were purchased from Sigma Chemical Co. (St. Louis, MO). [3H]thymidine and [{gamma}-32P]ATP were purchased from NEN Life Science Products, Inc. (Boston, MA). Enhanced chemiluminescence reagents, Hyperfilm ECL, and Hybond C nitrocellulose were purchased from Amersham Life Science (Arlington Heights, IL). Fyn antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Src antibodies were purchased from Oncogene Research Products (Cambridge, MA). Antibodies to Shc, IRS-1, MAPK-1/2 (ERK1/2-CT), and phosphotyrosine were purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies to dual phosphorylated ERK-1 and -2 MAPK were purchased from New England Biolabs (Beverly, MA). Yes antibodies were purchased from Santa Cruz Biotechnology, Upstate Biotechnology, and Wako Chemicals (Osaka, Japan). Human recombinant IGF-I was obtained from GroPep (Adelaide, Australia). Human recombinant EGF was purchased from Pepro Tech (Rocky Hill, NJ). PP1, PT, cholera toxin, and tyrphostin AG-1478 were purchased from BioMol (Plymouth Meeting, PA), and tyrphostin AG-1298 was from Calbiochem (La Jolla, CA). SRD/3T3 cells, which overexpress an activated form of Src (52) , and the plasmids containing wild-type c-Src and SrcRF were a gift from Dr. Joan Brugge (Harvard University, Cambridge, MA).

Cell Culture.
The murine preadipocyte line 3T3-L1 was obtained from American Type Culture Collection (Rockville, MD). Cells were grown in DMEM with L-glutamine, 1 g/liter glucose, 50 µg/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 10% fetal bovine serum. Cultures were maintained in an atmosphere of 5% CO2-95% humidified air at 37°C. SCM was replaced every 3 days. Proliferating cells were used for experiments when monolayers were 50–80% confluent, and cells were considered to be growth arrested 48 h after the monolayer was completely confluent.

Transient Transfection.
For transient transfection of 3T3-L1 cells, cells were seeded in 6-well plates and used at ~50% confluency. The plasmids, kindly provided by Dr. Joan Brugge (Harvard University), included wild-type c-Src or SrcRF cloned into the cytomegalovirus expression vector pCB6+. Wild-type c-Src or SrcRF DNA was transfected into cells at 1 µg/well using Gene Porter (Gene Therapy Systems) in serum-free Opti-MEM medium for 4 h at 37°C; then 20% SCM was added in a 1:1 volume overnight. Cotransfection of the plasmid pEGFP-F, which expresses a farnesylated green fluorescent protein that binds to plasma membranes in both living and fixed cells (Clontech, Palo Alto, CA), was used at 0.1 µg/well as a marker of transfection efficiency. The negative control was a mock transfection, i.e., the transfection protocol was performed, but no DNA was added except for pEGFP-F. The day after transfection, medium was changed to standard 10% SCM. Cells were used for analyses 24 h later, when they were still ~50% confluent.

Immunoprecipitation and Western Blot Analysis.
Preparation of total cellular lysates (500 µg) for immunoprecipitation and Western blotting was as described previously (15) using lysis buffer with 1% Triton X-100 for Shc and IRS-1 analyses or 0.2% Triton X-100 for MAPK analysis. Immunoprecipitation of Shc and IRS-1 was accomplished using protein A-Sepharose CL-4B beads (Pharmacia Biotech, Uppsala, Sweden) to which specific antibody had been covalently bound using dimethylpemilimidate. For immunoprecipitation of Src family members, 1 mg of total cellular lysates was harvested in RIPA buffer (without SDS) as described previously (35) , incubated with specific Src family member antibodies overnight at 4°C, and then immunoprecipitated with protein A-Sepharose beads for 2 h. Proteins were resolved by SDS-PAGE on 7.5% (IRS-1 immunoprecipitates) or 10% (Shc and SFK immunoprecipitates) acrylamide gels. For MAPK, 25 µg of total cell lysate protein were resolved on 10% acrylamide gels. Proteins were transferred to nitrocellulose or PVDF (IRS-1) membranes. Membranes were 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 was analyzed by digital image analysis using a Hewlett-Packard ScanJet 6100C/T scanner with Gel Pro Analyzer 3.1 software from Media Cybernetics.

[3H]Thymidine Incorporation.
For [3H]thymidine incorporation, cell monolayers were grown to ~50% confluence in 6-well plates and were serum starved overnight in DMEM with 0.1% BSA before treatment with 10 nM IGF-I for 24 h. Cells were also incubated with 10 µM PP1 (from a stock of 10 mM in DMSO) or an equal volume of DMSO for 24 h prior to the addition of 1 µCi/well [3H]thymidine for 4 h and then were lysed in 0.33 M NaOH. An aliquot was removed for protein assay prior to DNA precipitation with ice-cold 40% TCA/1.2 M HCl and collection on glass fiber filters for counting. The background level of [3H]thymidine was less than 200 cpm as determined by the addition of [3H]thymidine to a control well just before cell lysis.

Src Family Protein Kinase Assay.
An aliquot of SFK immunoprecipitates was removed for Western blot analysis. The remaining immunoprecipitates were washed in cold kinase buffer [50 mM PIPES (pH 7.0), 10 mM MnCl2, 10 mM DTT, and 200 µM sodium orthovanadate] and added to an in vitro kinase reaction consisting of kinase buffer, 1 µg of acid-denatured enolase, 10 µM ATP, and 5 µCi of [{gamma}-32P]ATP. Reaction mixtures were incubated at 30° for 5 min and were stopped by adding 5x sample buffer and boiling for 5 min. Src immunoprecipitated from 100 µg of total cell lysate protein from SRD/3T3 cells served as a positive control, and negative controls included RIPA buffer only and Src immunoprecipitated from IGF-I-stimulated cells, but no enolase. Proteins were separated on 10% polyacrylamide gels, washed in 7.5% acetic acid/7.5% methanol to reduce background, dried, and exposed to X-Omat film at -70°.


    Acknowledgments
 
We thank Dr. Joan Brugge for advice and Dr. Ed Filardo for assistance with experiments.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a Knoll Pharmaceutical Company Weight Risk Investigator Study Council Grant (to C. M. B.), NIH Grant HD24455 (to P. A. G.), and the Rhode Island Hospital Department of Pediatrics Research Endowment. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics, Rhode Island Hospital, 593 Eddy Street, MPS-2, Providence, RI 02903. Phone: (401) 444-7891; Fax: (401) 444-2534; E-mail: Charlotte_Boney{at}brown.edu Back

3 The abbreviations used are: IGF-I, insulin-like growth factor I; IGFR, type I IGF receptor; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; EGF, epidermal growth factor; SFK, Src family kinase; PDGF, platelet-derived growth factor; PT, pertussis toxin; SCM, serum-containing medium; ERK, extracellular regulated kinase. Back

Received for publication 1/11/01. Revision received 4/23/01. Accepted for publication 5/16/01.


    References
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 

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