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Endocrinology Vol. 144, No. 6 2546-2552
Copyright © 2003 by The Endocrine Society

C-Terminal Src Kinase (CSK) Modulates Insulin-Like Growth Factor-I Signaling through Src in 3T3-L1 Differentiation

Hiroko Sekimoto and Charlotte M. Boney

Department of Pediatrics, Brown Medical School and Rhode Island Hospital, Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Charlotte M. Boney, M.D., Department of Pediatrics, MPS-2, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: charlotte_boney{at}brown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I stimulates both proliferation and differentiation of adipocyte-precursor cells, preadipocytes in vivo and in vitro. We have previously shown that IGF-I stimulates proliferation of 3T3-L1 preadipocytes through activation of MAPK and MAPK activation by IGF-I is mediated through the Src family of nonreceptor tyrosine kinases. In addition, we have shown that when 3T3-L1 cells reach growth arrest and are stimulated to differentiate, IGF-I can no longer activate the MAPK pathway. We hypothesized that the loss of IGF-I signaling to MAPK in differentiating 3T3-L1 cells is due to loss of IGF-I activation of Src family kinases. We measured c-Src kinase activity in cell lysates from proliferating, growth-arrested and differentiating 3T3-L1 cells. Src activity increased 2- to 4-fold in IGF-I-stimulated proliferating cells; however, IGF-I had a marginal affect on Src activity in growth-arrested cells and inhibited Src activity localized at the membrane in differentiating cells. C-terminal Src kinase (CSK), a ubiquitously expressed nonreceptor tyrosine kinase, negatively regulates the Src family kinases by phosphorylation of the Src C-terminal tyrosine. IGF-I decreased phosphorylation of the Src C-terminal tyrosine in proliferating cells and increased phosphorylation of this site in differentiating cells. IGF-I stimulated CSK kinase activity 2-fold in differentiating 3T3-L1 cells. An association between CSK and c-Src was detected by immunoprecipitation following IGF-I stimulation of differentiating but not proliferating 3T3-L1 cells. These results suggest that the loss of IGF-I downstream mitogenic signaling in differentiating 3T3-L1 cells is due to a change in IGF-I activation of c-Src and CSK may mediate the inactivation of c-Src by IGF-I in 3T3-L1 adipogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF-I PLAYS AN IMPORTANT ROLE IN preadipocyte cell growth as well as differentiation to adipocytes in vitro and in vivo (1, 2, 3). The biological actions of IGF-I are mediated by its receptor tyrosine kinase, the type I IGF receptor (IGFR), which then activates various downstream signaling pathways. We have previously shown that IGF-I-stimulated mitogenesis in 3T3-L1 preadipocytes involves Shc phosphorylation that, in turn, mediates ERK-1 and -2 MAPK activation (4). The insulin receptor substrate proteins have been shown to mediate adipocyte differentiation in knockout mouse models and in vitro (5, 6). We have observed that IGF-I stimulation of Shc and MAPK, but not insulin receptor substrate-1, is lost when density-induced, growth-arrested 3T3-L1 preadipocytes are stimulated to differentiate (7). This loss of IGF-I mitogenic signaling suggests modulation in the pathway from the IGFR to MAPK. More recently we demonstrated that IGF-I activates members of the Src family of nonreceptor tyrosine kinases and Src kinases mediate IGF-I-stimulated phosphorylation of Shc and activation of the downstream MAPK pathway in proliferating 3T3-L1 preadipocytes (8).

It is now well established that Src family kinases play important roles in normal cell growth. Src family kinases mediate mitogenesis by several growth factors through activation of the Shc-MAPK signaling pathway (9, 10). Regulation of Src family kinase activity is known to occur through a number of mechanisms, including subcellular localization and phosphorylation. Src kinases are recruited to cell membranes and activated by signaling pathways downstream from growth factors and integrins (11). An important mechanism of Src family kinase regulation is phosphorylation/dephosphorylation of critical Src tyrosine (Y) residues (12). Src phosphorylation at Y-418 in the murine protein (human Y-419), which is the autophosphorylation site in the kinase domain, is required for full activation. Autophosphorylation at this site occurs following binding of Src homology (SH)2 and SH3 domain-containing proteins, such as tyrosine kinase receptors and various signaling molecules.

The best-characterized negative regulator of Src family kinases is C-terminal Src kinase (CSK), a ubiquitously expressed nonreceptor tyrosine kinase that phosphorylates the Src family kinase C-terminal Y-529 (Y-530 in humans) (13, 14, 15). Phosphorylation at Y-529 leads to its intramolecular binding to the Src SH2 domain, resulting in folding and inactivation of the Src family kinase. CSK has been previously demonstrated to play a role in IGF-I inhibition of c-Src (16). In the present study, we observed that IGF-I activation of membrane associated-Src changes as 3T3-L1 cells undergo differentiation and CSK likely modulates the inactivation of c-Src by IGF-I in 3T3-L1 adipogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Buffer reagents, enolase, and CSK peptide substrate were purchased from Sigma (St. Louis, MO) and X-Omat AR film from Kodak (Rochester, NY). {gamma}-32P-ATP was purchased from NEN Life Science Products (Boston, MA). Enhanced chemiluminescence (ECL) reagents and hyperfilm ECL were purchased from Amersham Life Science (Arlington Heights, IL). Src antibodies for Western blotting were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Src antibodies for immunoprecipitation and SRD/3T3 cells, which overexpress an activated form of Src (17), were generous gifts from Dr. Joan Brugge (Harvard University, Boston, MA). Phospho-specific Src antibodies (anti-Src[pY418] and anti-Src[pY529]) were purchased from Biosource Technologies, Inc. (Camarillo, CA). CSK antibodies for immunoprecipitation were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and CSK antibodies for Western blotting were purchased from BD Transduction Laboratories, Inc. (Lexington, KY). IGFR antibodies were generously provided by Dr. Robert J. Smith (Brown Medical School, Providence, RI). Human recombinant IGF-I was obtained from GroPep (Adelaide, Australia).

Cell culture
The murine preadipocyte line 3T3-L1 was obtained from American Type Culture Collection (Manassas, VA). 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. Serum-containing medium (SCM) was replaced every 3 d. Proliferating cells were used for experiments when monolayers were 50–80% confluent. Cells were considered to be growth arrested 48 h after the monolayer was completely confluent. Postconfluent, growth-arrested cells were stimulated to differentiate with a cocktail of 0.5 µM dexamethasone, 0.5 mM methylisobutylxanthine, and 10 nM IGF-I in SCM. The medium was changed 48 h later to SCM, and differentiating cells were then harvested 72 h later when more than 75% of the population was adipocytes. Monolayers were placed in serum-free medium with 0.1% fatty acid-free BSA (Sigma) overnight before experiments the next day.

Preparation of subcellular fractions
Total cell lysates were fractionated into nuclear, cytosolic, and membrane components based on the procedure by Joost and Schurmann (18). Monolayers were washed in Tris-EDTA-sucrose (TES) buffer without protease inhibitors (20 mM Tris-HCl, pH 7.4; 1 mM EDTA; 8.7% sucrose; 1 mM Na3VO4) and then lysed in TES buffer with 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride. Total cell lysate protein was determined by bicinchoninic assay protein assay (Pierce Chemical Co., Rockford, IL), and 5 mg total protein were homogenized and centrifuged at 8000 x g for 5 min. The pellet containing the nuclear fraction was resuspended in TES buffer containing 1% Nonidet P-40 layered onto 30% sucrose in TES buffer and centrifuged at 2500 rpm for 5 min. The pellet was resuspended in modified Tris-buffered saline (TBST) (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X 100, 1 mM Na3VO4, and protease inhibitors), sonicated, and centrifuged at 13,000 rpm for 20 min. The supernatant was collected as the nuclear fraction. Supernatant from the first low-speed centrifugation step, which contained the cytosol and membrane fractions, was centrifuged at 30,000 x g for 30 min. The supernatant was set aside as the cytosol fraction, and the pellet was resuspended in TBS, sonicated, and centrifuged at 13,000 rpm for 20 min, with the resulting supernatant containing the membrane fraction. The purity of fractions was determined by analyzing nuclear and soluble fractions for glucose-6-phosphate dehydrogenase activity as a marker of the cytosol and 5'-nucleotidase (5'ND) activity as a marker of the membrane (kits from Sigma). Glucose-6-phosphate dehydrogenase activity was 0.30 ± 0.08 U/mg in cytosol and <0.05 U/mg in nuclear and membrane fractions, whereas 5'ND activity was 20 ± 5 mU/mg in membrane and less than 5 mU/mg in nuclear and cytosol fractions. The lower limit of detection of the 5'ND assay is 5 mU/mg; therefore, we cannot completely rule a low level of contamination among the subcellular preparations.

Immunoprecipitation and Western blot analysis
Cell monolayers were harvested in RIPA assay buffer (without SDS) (19) for Src analysis and then total cell lysate protein determined by bicinchoninic assay protein assay (Pierce Chemical Co.). For immunoprecipitation (IP) of Src, 1 mg total protein was incubated with primary antibodies for 4 h at 4 C. For IP of IGFR, 2 mg total protein were incubated with specific antibodies for 4 h at 4 C. Nuclear, cytosolic, or membrane fractions were immunoprecipitated with primary antibody for 4 h at 4 C. Protein A-Sepharose was then added for 2 h to precipitate immune complexes. Immunoprecipitated proteins were used immediately in kinase assays or resolved by SDS-PAGE on 10% acrylamide gels and transferred to polyvinyl difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA). Membranes were blocked in 5% BSA in TBST with 0.1% Triton X-100 for exposure to phospho-specific antibodies and phosphotyrosine antibodies; 5% nonfat dry milk in TBST was used for blocking for total Src and IGFR antibodies. Primary antibody was generally used at 1 µg/ml in TBST and secondary antibody at 0.5 µg/ml in TBST. Specific binding was visualized using ECL and Hyperfilm ECL and then analyzed by digital image analysis using a ScanJet 5470c scanner (Hewlett-Packard Co., Palo Alto, CA) with Gel Pro Analyzer 3.0 software (Media Cybernetics, Silver Spring, MD).

Src kinase assay
An aliquot of immunoprecipitated c-Src was removed for Western blot analysis of total Src content, and the remaining immunoprecipitated c-Src was assayed in an in vitro kinase assay using enolase as the substrate as described previously (8). Constitutively active c-Src immunoprecipitated from 100 µg total cell lysate protein from SRD/3T3 cells served as a positive control, and negative controls included buffer only and sample 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 (Kodak) at -70 C. Enolase autoradiograms were quantified by densitometry as described above.

CSK kinase assay
This kinase assay was modified from Sondhi et al. (20). Monolayers were harvested in modified TBST, and an aliquot of was removed for measurement of total protein content. One milligram protein of fresh lysates was immunoprecipitated using anti-CSK antibodies (Santa Cruz Biotechnology, Inc.) or nonspecific IgG for 2 h at 4 C followed by incubation with protein-A Sepharose beads for 1 h. The immunoprecipitates were washed in modified TBST, and an aliquot was removed for Western blotting to determine CSK recovery. The immunoprecipitates were washed in kinase buffer consisting of 60 mM Tris-HCl, pH 7.4, 5 mM MnCl2, 10 mM dithiothreitol, and 200 µM Na3VO4 and resuspended in 15 µl kinase buffer. Reaction mixture consisting of 1 µCi [{gamma}-32P]ATP, 500 µM cold ATP, and 200 µM CSK peptide substrate KKKKEEIYFFF (Sigma), previously shown by Sondhi et al. (20) to be highly specific for CSK, was added to each tube and incubated 5 min at 28 C. In addition to immunoprecipitates using nonspecific IgG, a negative control included sample but no substrate. The reaction was stopped by adding 10 µl 250 mM EDTA. The reaction mixture was centrifuged and the supernatant spotted onto p81 phosphocellulose filters (Whatman International, Maidstone, UK). The filters were washed extensively in 75 mM phosphoric acid and counted in a scintillation counter (Tri-Carb 2000CA, Packard Instrument Co., Downers Grove, IL). Negative controls had the same low activity as background (200–300 cpm), so these counts were not subtracted from the true samples. Activity was calculated as nanomoles 32P incorporated into milligrams protein substrate per minute.

Statistical analysis
Unless otherwise noted, ANOVA was used to determine differences found in experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I-stimulated c-Src activity changes as 3T3-L1 cells progress from proliferating preadipocytes to differentiating adipocytes
It is well described in in vitro models of adipogenesis that IGF-I stimulates proliferation of preadipocytes, but when cells reach density-induced growth arrest, IGF-I in the presence of a differentiation cocktail stimulates differentiation (1). We have previously shown that Shc phosphorylation and downstream MAPK activation by IGF-I is mediated through c-Src (8) and IGF-I-activated Shc and MAPK is dramatically decreased in growth-arrested 3T3-L1 preadipocytes stimulated to differentiate (7). We hypothesized that the loss of IGF-I signaling to MAPK in differentiating 3T3-L1 cells is due to loss of IGF-I activation of c-Src. To determine whether IGF-I activation of c-Src changes as 3T3-L1 cells progress from proliferating preadipocytes to differentiating adipocytes, we measured the kinase activity of c-Src in cell lysates using an in vitro kinase assay in which enolase is the substrate (Fig. 1AGo). IGF-I stimulated c-Src kinase activity 4-fold in proliferating cells; however, IGF-I stimulation of c-Src in growth-arrested cells was decreased to approximately 30% above basal, and IGF-I inhibited c-Src activity in differentiating cells. The striking change in c-Src kinase activity as 3T3-L1 cells progressed from proliferating to growth arrested to differentiating was in basal c-Src activity. This dramatic increase in basal c-Src activity accounts for the decrease in fold stimulation by IGF-I in growth-arrested cells, and the high basal activity was preserved in differentiating 3T3-L1 cells.



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  		Figure 1. IGF-I activation of c-Src from proliferating (P), growth-arrested (GA), and differentiating (D) 3T3-L1 cells. Cells were serum starved overnight and then treated with 10 nM IGF-I for 1 min (solid bars) or left untreated (open bars) before cell lysis and immunoprecipitation with anti-Src antibodies. A, Kinase activity of Src IPs was measured in an assay using enolase as substrate, and then autoradiograms were quantified by densitometry. Graph shows the mean + SD of six independently performed kinase assays. ***, P < 0.001; *, P < 0.02. B, Western blot analysis (left) of Src IP from P, GA, and D cells using anti-Src-PY-418 to detect the active form of Src. Blots were stripped and probed with anti-Src to determine total Src protein (not shown). Corresponding densitometry results (right) for PY-418 content are corrected for total Src content. ***, P < 0.001; *, P < 0.05.

 
To confirm the observation made from the kinase assay, we assessed c-Src activity indirectly using Western blot analysis with specific phospho-Src antibodies (Fig. 1BGo). These antibodies are selective for the active form of Src containing phosphotyrosine (PY) at position 418, the autophosphorylation site located in the kinase domain that is required for full catalytic activity of Src family kinases. The same pattern of Src activation was observed: IGF-I increased the abundance of PY-418 in proliferating cells and modestly decreased PY-418 abundance in differentiating cells. In addition, the high basal levels of PY-418 in growth-arrested and differentiating cells was confirmed, consistent with high basal c-Src activity. Of note, total c-Src protein recovered in the immunoprecipitates was not different among the three cell groups (data not shown).

The shift in IGF-I-stimulated to IGF-I-inhibited c-Src activity occurs mostly in c-Src localized at the membrane
Subcellular localization of c-Src is thought to play a major role in its regulation and function (12). For example, c-Src localized at the plasma membrane has been shown to mediate mitogenic signaling events downstream of growth factor receptors (9). Therefore, we fractionated total cell lysates into nuclear, cytosolic, and membrane components to identify the subpopulation of c-Src being regulated by IGF-I and the subpopulation of c-Src with high basal activity in growth-arrested and differentiating cells (Fig. 2Go). c-Src was immunoprecipitated from subcellular fractions, and then Western blot analysis using Src-PY-418 antibodies was performed to indirectly assess c-Src activity. IGF-I dramatically increased PY-418 in the membrane fraction of proliferating cells, consistent with potent activation of c-Src by IGF-I. IGF-I-stimulated PY-418 was dramatically decreased in c-Src from membranes of growth-arrested cells. IGF-I decreased the abundance of PY-418 in differentiating cells, consistent with IGF-I inhibition of c-Src in the membrane fraction. This shift in IGF-I-stimulated phosphorylation of PY-418 in membrane fractions was similar to the pattern of Src kinase activity observed in total cell lysates.



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  		Figure 2. IGF-I activation of c-Src from subcellular fractions of proliferating, growth-arrested, and differentiating cells. Cells were serum starved overnight and then treated with 10 nM IGF-I for 1 min (closed bars) or left untreated (open bars) before cell lysis. Five milligrams of total cell lysate protein were subfractionated into membrane, cytosol, and nuclear fractions. Src was immunoprecipitated, and Western analysis was done using anti-Src-PY-418 to indirectly assess activity. Blots were stripped and probed with anti-Src to determine total Src protein. Results are shown as densitometry of PY-418 corrected for total Src protein for each cell stage (mean + SD, n = 3; ***, P < 0.001; *, P < 0.05).

 
IGF-I had a small but significant stimulatory effect on PY-418 abundance, and thus c-Src activity, in the cytosol and nucleus of proliferating cells. IGF-I did not significantly affect cytosolic c-Src PY-418 in growth-arrested and differentiating cells (Fig. 2Go). The high basal levels of PY-418 seen in growth-arrested and differentiating cells was localized to the nucleus, and IGF-I had no effect on these nuclear fractions of Src. Western blots of total Src revealed twice the amount of c-Src in nuclear fractions, compared with membrane or cytosol fractions, and this distribution of Src did not change with IGF-I treatment, and it was not different among proliferating, growth-arrested, or differentiating cells (data not shown). There was no change in the proportion of nuclear Src recovered from the immunoprecipitations in growth-arrested and differentiating cells, suggesting the high Src activity in the nucleus is not likely due to a change in Src distribution. These results indicate that the subfraction of c-Src regulated by IGF-I is mostly localized to the membrane and the high basal, IGF-I-independent Src activity in growth-arrested and differentiating cells is in the nucleus.

IGF-I inhibition of c-Src activity in differentiating 3T3-L1 adipocytes is associated with phosphorylation of c-Src C-terminal tyrosine
To investigate the switch in IGF-I signaling from activation to inhibition of c-Src located at the membrane, we determined the phosphorylation state of the critical Y at position 529 involved in inhibition of c-Src activity. c-Src was immunoprecipitated from membrane fractions of proliferating, growth-arrested, and differentiating cells, and phosphorylation of Y-529 was determined by phospho-specific Western blotting (Fig. 3Go). Basal PY-529 was readily detectable and not significantly different among proliferating, growth-arrested, and differentiating cells; however, IGF-I decreased PY-529 in proliferating cells, consistent with Src activation but increased PY-529 in differentiating cells, consistent with Src inhibition.



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  		Figure 3. IGF-I phosphorylation of Src C-terminal Y from proliferating, growth-arrested, and differentiating 3T3-L1 cells. Cells were serum starved overnight and then treated with 10 nM IGF-I for 1 min or left untreated before cell lysis and subfractionation. The c-Src was immunoprecipitated from membrane fractions, and then Western blot analysis was performed using anti-Src-PY-529 to identify the inactive form of Src. This was followed by Western analysis for total Src protein (not shown). A representative Western blot of PY-529 is shown. Densitometry results of PY-529 corrected for total Src protein compiled from two experiments are shown (mean + SD, n = 4; *, P < 0.05; **, P < 0.01).

 
CSK inactivates Src family kinases via phosphorylation of Y-529 (21). Given the increase in PY-529 observed with IGF-I stimulation as the cells undergo differentiation, it was important to determine whether CSK was also activated by IGF-I. This was accomplished by immunoprecipitating CSK from proliferating and differentiating 3T3-L1 cells and measuring its kinase activity in an in vitro kinase assay (Fig. 4Go). IGF-I had little effect on CSK activity in proliferating cells, but IGF-I increased CSK kinase activity 2-fold in differentiating cells. These data indicate a parallel increase in tyrosine phosphorylation at position 529, the CSK site, and CSK kinase activity in differentiating cells, suggesting a CSK-dependent mechanism for IGF-I-mediated Src inactivation.



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  		Figure 4. IGF-I activation of CSK activity in proliferating and differentiating 3T3-L1 cells. In separate experiments, proliferating or differentiating cells were serum starved overnight and then treated with 10 nM IGF-I for 1 min. Cell lysates containing 1 mg total protein were immunoprecipitated with anti-CSK antibodies, and then activity was measured in an in vitro kinase assay. Equivalent amounts of CSK were confirmed by Western blotting (data not shown). Activity is expressed as 32P incorporation into peptide substrate (nanomoles per minute per milligram protein; mean + SD, n = 3; *, P < 0.02 by t test).

 
IGF-I promotes the association of c-Src with CSK in differentiating 3T3-L1 adipocytes
Arbet-Engels et al. (16) have previously shown that IGF-I inhibits c-Src through an association of the IGFR with CSK in human 293 cells and NIH 3T3 fibroblasts. We therefore tested for a direct association between the IGFR and CSK or between the IGFR and Src in 3T3-L1 cells by coimmunoprecipitation. The IGFR was immunoprecipitated from 2 mg total cell lysate protein from proliferating and differentiating cells, and then Western blots were performed. Although we could easily observe an increase in PY of the IGFR with IGF-I stimulation, we could not detect c-Src or CSK in the IGFR immunoprecipitates from either proliferating or differentiating cells (data not shown). This may indicate that in 3T3-L1 cells, the IGFR does not directly activate c-Src or CSK, or it may be that the interaction between IGFR and c-Src or CSK is not preserved following cell lysis and thus cannot be detected by this method.

Although we could not demonstrate a direct association between the IGFR and CSK, we investigated the association of CSK and c-Src in IGF-I-stimulated 3T3-L1 cells. CSK was immunoprecipitated from proliferating and differentiating cells, and then Western blot analysis was performed with anti-CSK and anti-Src antibodies (Fig. 5Go). Protein abundance of CSK was equivalent in proliferating and differentiating cells, indicating no change in CSK expression. There was minimal c-Src associated with CSK in the basal state, but coimmunoprecipitation of Src with CSK was detectable in IGF-I-stimulated differentiating 3T3-L1 cells. The results of two other similar experiments support the conclusions drawn from the experiment shown in Fig. 5Go. The reproducibility of c-Src-associated CSK was dependent on IP of fresh, not frozen, cell lysates using short incubation periods with anti-CSK antibody, consistent with a rapidly reversible association between CSK and c-Src.



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  		Figure 5. Effect of IGF-I on the association of c-Src and CSK in 3T3-L1 cells. Proliferating and differentiating cells were serum starved overnight followed by stimulation with 10 nM IGF-I for min. Fresh cell lysates containing 2 mg total protein were immunoprecipitated with anti-CSK antibodies and then analyzed by Western analysis using anti-Src antibodies. Membranes were then stripped and reprobed with anti-CSK antibodies. Negative controls included IP of IGF-I-stimulated lysates with nonimmune IgG and incubation of IGF-I-stimulated lysates with protein A Sepharose alone without antibody (not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that IGF-I-stimulated c-Src activity parallels the changes seen in IGF-I downstream mitogenic signaling in 3T3-L1 cells undergoing differentiation. Our present results indicate that IGF-I activation of Src decreases in growth-arrested cells and is actually inhibited in differentiating cells and this change in IGF-I-regulated Src activity is restricted to the membrane. This is not surprising, given that Src located at the membrane is well described to mediate the mitogenic effect of growth factors (22). Src family kinases are localized to cell membranes via myristylation and specific basic residues at the amino terminus (23, 24). In fibroblasts, Src family kinase members are recruited to the membrane following activation (25).

Indirect measurement of Src activity (i.e. state of phosphorylation on Y-418) indicates that IGF-I treatment for 1 min has a minimal effect on c-Src activity in the cytosol and nucleus of 3T3-L1 cells, although there may be a small effect in proliferating cells. Initial time course experiments of Src activation were performed in whole-cell lysates of proliferating cells in which IGF-I activation of the MAPK pathway is highest (8), so we did not repeat time course experiments to determine maximal IGF-I activation of Src in the other stages. Therefore, it is possible that the kinetic activation of Src by IGF-I is different (i.e. later than 1 min) in growth-arrested and differentiating cells. However, delayed activation of Src by IGF-I in these stages, although interesting, would have a function unrelated to IGF-I activation of the MAPK pathway because IGF-I activation of MAPK is low in growth-arrested cells and negligible in differentiating cells.

The function of increased basal Src activity, independent of IGF-I, in the nucleus of growth-arrested and differentiating 3T3-L1 cells is unknown but may indicate cell cycle regulation that has been shown to occur by Src family kinase members in other cell types (26, 27, 28). The high basal activity probably is not due to translocation of activated Src into the nucleus because distribution of total Src protein did not change; however, recovery of Src measured by Western blot following IP may not be sensitive enough to detect small changes in Src redistribution.

The mechanism of IGF-I activation of c-Src in 3T3-L1 cells remains unknown. In the present study, we could not detect an association of the IGFR with Src by immunoprecipitation, but this does not definitively prove that Src is not directly activated by the IGFR. Other mitogens have been shown to activate Src family kinases directly through SH2 binding of the kinase to the receptor tyrosine kinase (9). Growth factors have also been shown to activate Src family kinases indirectly through transactivation of other receptors including G protein-coupled receptors (29). We have previously shown that IGF-I activation of c-Src does not involve transactivation of the epithelial growth factor receptor or heterotrimeric G proteins (8). Growth factor receptor interactions with integrins or complex signaling pathways that affect Src family kinase phosphorylation and dephosphorylation are well described and more likely mediators of IGF-I action (11, 30).

An important mechanism for regulation of Src family kinase activity is the phosphorylation status of two critical Y residues, Y-418 and Y-529 (12). In addition to the CSK-mediated inhibition through phosphorylation of Y-529, a number of protein tyrosine phosphatases (PTPs) have been demonstrated to activate Src family kinases by dephosphorylation of PY-529 (12, 31, 32). Therefore, kinase activity depends in part on a balance between dephosphorylation and phosphorylation of Y-529.

We observed that c-Src activation by IGF-I in 3T3-L1 cells parallels a change in the phosphorylation of Y-529 as cells undergo differentiation. We have shown that the change from IGF-I-stimulated to IGF-I-inhibited c-Src is associated with an increase in PY-529 and CSK activity. In proliferating 3T3-L1 cells, IGF-I increases PY-418 and decreases PY-529, consistent with Src activation. However, IGF-I does not inhibit CSK activity in proliferating cells, so we can only speculate that IGF-I may activate a PTP that is responsible for the decrease in PY-529. In differentiating cells, IGF-I increases the association of CSK and Src and stimulates CSK activity, suggesting that IGF-I inactivation of c-Src is mediated by CSK phosphorylation of Y-529. Others have shown that PTP{alpha}, a ubiquitously expressed receptor-like PTP, positively regulates Src activity in 3T3-L1 adipocytes (31). Therefore, it is possible that the increase in PY-529 may reflect IGF-I-inhibition of PTP{alpha}, but our data support a role for CSK in IGF-I signaling in 3T3-L1 adipocytes.

CSK regulation is not well understood, but relocalization of CSK by one or more CSK-binding proteins may play a role in its activation and subsequent inhibition of Src family kinases (33). There is increasing evidence that other signaling proteins, such as PTPs (34), cytoskeletal components (35), integrins (36), and specific adaptor proteins containing SH2 and SH3 domains (37) recruit CSK to various subcellular sites to regulate Src family kinase activity. CSK activity does not appear to be regulated by IGF-I in proliferating cells, but it still may play a role in maintaining basal Src in the inactive form. When 3T3-L1 cells undergo differentiation, IGF-I is able to activate CSK through an unidentified mechanism. We could not detect a direct association between the activated IGFR and CSK, although that association may be of low affinity, easily reversible, and not detectable by coimmunoprecipitation. Alternatively, our data may reflect a change in CSK regulation that results from recruitment by IGF-I to Src-containing signaling complexes and inhibition of Src-mediated mitogenic pathways during differentiation.

In summary, we have demonstrated that IGF-I activation of membrane-associated c-Src changes as 3T3-L1 preadipocytes progress from proliferation to differentiation, consistent with earlier observations that loss of IGF-I mitogenic signaling is a component of IGF-I-stimulated adipogenesis (Fig. 6Go). In addition, IGF-I inactivation of c-Src in 3T3-L1 adipocytes is associated with increased CSK activity, consistent with previously published models of Src regulation through recruitment of CSK to Src signaling complexes (38). We speculate that IGF-I regulation of c-Src activity in 3T3-L1 cells is mediated through a dynamic signaling complex that changes during adipogenesis.



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  		Figure 6. Proposed model of IGF-I signaling through Src in proliferating and differentiating 3T3-L1 cells. Our previously published data and the present results support a model of IGF-I signaling through c-Src to MAPK in proliferating preadipocytes, although we have not identified the mechanism of IGF-I activation of c-Src. When growth-arrested cells are stimulated to differentiate, IGF-I inhibits Src and can no longer activate the downstream Shc-MAPK pathway, thus allowing differentiation to proceed. The mechanism of Src inhibition appears to involve IGF-I activation of CSK, likely through a large signaling complex.

 


   Acknowledgments
 
We thank Dr. Barbara Giovannone for technical advice and Dr. Robert J. Smith and Dr. Philip Gruppuso for helpful discussions and advice.


   Footnotes
 
This work was supported by NIH Grant RO1-DK-59339 and Rhode Island Hospital Department of Pediatrics Research Endowment.

Abbreviations: CSK, C-terminal Src kinase; ECL, enhanced chemiluminescence; IGFR, IGF receptor; IP, immunoprecipitation; 5'ND, 5'-nucleotidase; PTP, protein tyrosine phosphatase; PY, phosphotyrosine; SCM, serum-containing medium; SH, Src homology; TBST, Tris-buffered saline; TES, Tris-EDTA-sucrose; Y, tyrosine.

Received February 10, 2003.

Accepted for publication March 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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