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Endocrinology Vol. 142, No. 5 1835-1840
Copyright © 2001 by The Endocrine Society

ARTICLES

The Insulin-Like Growth Factor I Receptor-Induced Interaction of Insulin Receptor Substrate-4 and Crk-II

M. Karas1, A. P. Koval1,2, Y. Zick and D. LeRoith

Clinical Endocrinology Branch (M.K., A.P.K., D.L.), NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1758; and Department of Molecular and Cell Biology (Y.Z.), The Weizmann Institute of Science, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Derek LeRoith, NIH, NIDDK, CEB, Building 10/Room 8D12, 10 Center Drive, MSC 1758, Bethesda, Maryland 20892-1758. E-mail: derek{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stimulation of the insulin or insulin-like growth factor (IGF)-I receptor results in activation of several signaling pathways. Proteins of the insulin receptor substrate (IRS) family play important roles in mediating these signaling cascades. To date, four members of the IRS family of docking proteins have been characterized. Recently, we have reported that stimulation of the IGF-I receptor in 293 HEK cells regulates interaction of the newly discovered IRS-4 molecule with the Crk family of proteins. In the present study, we characterize the molecular basis of these interactions. C- and N termini truncation analysis of IRS-4 demonstrated that the region between amino acids 678 and 800 of the IRS-4 molecule is involved in this interaction. This region contains a cluster of four tyrosines (Y700, Y717, Y743, and Y779). We hypothesize that one or more of these tyrosines are involved in the interaction between the SH2 domain of the Crk-II molecule when IRS-4 is phosphorylated upon IGF-I receptor activation. Additional mutational analyses confirmed this hypothesis. Interestingly, none of these four tyrosines was individually critical for the interaction between Crk-II and IRS-4, but when all four tyrosines were simultaneously mutated to phenylalanine, the IGF-I induced interaction between these molecules was abolished. Taken together, these results suggest a novel mechanism of Crk-II binding to tyrosine phosphorylated proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN AND insulin-like growth factor I (IGF-I) receptors are tyrosine kinase receptors that share a high degree of similarity, particularly in the tyrosine kinase domain (1). Both receptors share not only structural and sequence homology, but also a number of downstream targets, including Shc, PI3'-kinase, and the IRS family of proteins. The IRS proteins function as docking proteins, providing an interface between the activated insulin and IGF-I receptors and downstream SH2 domain-containing targets (2). To date, four IRS molecules have been characterized (3). At the N terminus, each IRS family member possesses a pleckstrin homology (PH) domain followed by a phosphotyrosine binding (PTB) domain (2). Multiple tyrosine phosphorylation sites are located throughout the remaining portion of the molecule. The role of IRS-1 and IRS-2, the first two members of the IRS family to be characterized, in insulin and IGF-I signal transduction has been extensively studied. IRS-1 and IRS-2 have been shown to interact with multiple proteins, including the 85-kDa subunit of PI3'-kinase, Grb-2, Crk, Nck, and others (2). However, the role of IRS-3 and IRS-4 in IGF-I and insulin receptor signal transduction of is far less clear, especially because the gene-deletion IRS-3 and IRS-4 mice failed to demonstrate a definitive phenotype (4, 5). Recently, we demonstrated that in HEK 293 cells, IRS-4 interacts with Crk-II and CrkL upon IGF-I receptor stimulation (6). Crk-II and CrkL are adapter proteins consisting primarily of SH2 and SH3 domains, which mediate certain protein-protein interactions. These proteins have been shown to be involved in signaling pathways that lead to cell growth, cytoskeletal rearrangement, differentiation, apoptosis, and transformation (7). In the present study, we have characterized the molecular mechanisms underlying the IGF-I receptor induced interaction of IRS-4 with Crk-II.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfections
Both 293 HEK and WTB3 NIH-3T3 mouse fibroblasts overexpressing the IGF-I receptor (8) were cultured in DMEM (Biofluids, Inc., Rockville, MD) supplemented with 10% FBS (Upstate Biotechnology, Inc., Lake Placid, NY). WTB3 cells were transiently transfected with plasmids encoding either full length, truncated or mutated hIRS-4. For these experiments, cells were plated in 100-mm dishes, and transfections were performed using Lipofectamine Plus reagent (Life Technologies, Inc., Rockville, MD). Transfected cells were allowed to recover for 24 h in complete medium, before switching to serum-free DMEM supplemented with 0.5% BSA. Cells were stimulated in the presence or absence of 50 nM IGF-I for 5 min. Stimulated and nonstimulated cells were then washed with ice-cold PBS and proteins were extracted with 1 ml of lysis buffer containing 20 mM Tris-HCl pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 10 mM sodium pyrophosphate, 2.5 mM EDTA, 2 mM sodium orthovanadate, 0.3 mM sodium molybdate, 10 mM sodium fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 10 µg/ml trypsin inhibitor. Cell lysates were then incubated for 10 min at 4 C and the nonsoluble fraction was removed by centrifugation (13,000 x g for 30 min at 4 C). The protein content was determined using the BCA protein assay reagent (Pierce Chemical Co., Rockford, IL).

Cloning of hIRS-4
Different fragments of hIRS-4 complementary DNA (cDNA) (primers and cloning strategy are available upon request) were amplified from the total RNA or genomic DNA purified from 293 cells. Full-length IRS-4 cDNA was obtained by combining appropriate fragments into pTrcHisB vector (Invitrogen, Carlsbad, CA). Both strands of the resulting construct encoding full-length hIRS-4 were sequenced and were found to be identical to the previously published sequence of IRS-4 (3). The full-length and N terminus-truncated variants 1–315 amino acids (aa), 1–678 aa and 1–1140 aa of IRS-4 were then subcloned into the eukaryotic expression vector pCEFL KZ HA (kindly provided by Dr. J. S. Gutkind, NIH, Bethesda, MD) (see Fig. 2AGo). To express C-terminal truncated variants of IRS-4, full length IRS-4 cDNA was digested with either BglII, SacI, or BsmI, to generate 976-1257aa, 801-1257 aa, and 678-1257 aa, respectively. These fragments were then religated back into the pCEFL KZ HA vector (Fig. 2AGo).



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  		Figure 2. Interaction of IRS-4 truncation mutants with Crk-II-GST. A, Schematic representation of N- and C-terminal deletion mutants of hIRS-4. Full-length hIRS-4 is indicated as FL. Pleckstrin homology and phosphotyrosine binding domains indicated as PH and PTB, respectively. The potential sites of tyrosine phosphorylation (3 ) are indicated. B, Interaction of Crk-II with N- and C-terminal deletion mutants of hIRS-4. WTB3 cells were transiently transfected with the indicated constructs and incubated in the presence or absence of 50 nM IGF-I for 5 min. Whole cell lysates were then pulled down with Crk-II-GST as described in Materials and Methods. The resulting associated proteins were subjected to SDS-PAGE followed by immunoblotting with antiphosphotyrosine antibodies.

 
Mutagenesis
Site directed mutagenesis was used to change Y700, Y717, Y743, and Y779 to F700, F717, F743, and F779. These amino acids are all localized within the BsmI-BglII fragment (nt 2080–3006), and the mutagenesis was performed as described previously (9). Universal flanking primers were:

5'-Bsm: 5'-TTGTGTTGACAGAGGAGCCACGA-3' (2049–2071)

3'-Bgl: 5'- GGTTGGCACGTGGTATAGCTCT-3' (3037–3017)

Primers for the Y770->F770 mutation were:

dir: 5'-CCCATTCGTGCCAATGAGGCCA-3'

rev: 5'-TTGGCACGAATGGGTCATCCTCA-3'

Primers for the Y717->F717 mutation were:

dir: 5'-GTGATTTTATTGCCAATGGCTCCT-3'

rev: 5'-TTGGCATAAAATCACTGGAGCTTA-3'

Primers for the Y743->F743 mutation were:

dir: 5'-GAGGGTTCATGATGATGTTTCC-3'

rev: 5'-TCATCATGAACCCTCTTGAATCTTC-3'

Primers for the Y779->F779 mutation were:

dir: 5'-GTGACTTCATGTTTATGGCTCCT-3'

rev: 5'-TAAACATGAAGTCACTCTCACTGT-3'

After the creation of individual mutations, the resulting BsmI-BglII fragment was inserted into the pB42AD vector in place of the analogous fragment encoding the wild-type protein. After confirmation of the mutations by DNA sequencing, the resulting full-length mutated IRS-4 was subcloned into the pCEFL KZ HA vector. Double mutants Y700Y717->F700F717 and Y743Y779->F743F779 were constructed in a similar procedure using single mutants as templates. To obtain all four mutated tyrosines in a single construct, BsmI-HaeII and HaeII-BglII fragments carrying double mutations were coligated.

IRS-4 interactions with GST fusion proteins
GST pulldown experiments were performed as previously described with minor modifications (6). Briefly, 200 µg of protein from cell lysates in a total volume of 500 µl of lysis buffer were mixed with 5 µg of SH-2-SH-3-containig Crk-II-GST fusion protein and 50 µl of a 50% (wt/vol) suspension of glutathione-Sepharose 4B beads in lysing buffer and were incubated overnight at 4 C. Samples were then washed twice with 1 ml of ice-cold lysis buffer. Precipitated samples were boiled for 5 min in loading buffer, fractionated by SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis.

Immunopreciptations
Immmunopreciptation experiments were performed as previously described with minor modifications (6). Briefly, 200 µg of protein from cell lysates in a total volume of 500 µl of lysis buffer were mixed with 5 µl of IRS-4 antiserum and were incubated overnight at 4 C. Fifty microliters of a 50% (wt/vol) suspension of protein A-Sepharose beads in lysis buffer was added and the incubation was continued for an additional 4 h. Samples were then washed twice with 1 ml of ice-cold lysis buffer. Precipitated samples were boiled for 5 min in loading buffer, fractionated by SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis.

Generation of anti-IRS-4 antibodies
To study the role of IRS-4 in IGF-I receptor signaling we generated rabbit polyclonal antibodies against a peptide encoding the last 16 C-terminal amino acids of the hIRS-4 protein. Whole serum from the immunized rabbit was tested in Western blots using cell extracts from 293 HEK cells expressing high levels of IRS-4 (3) and compared with polyclonal anti-hIRS-4 antibodies developed in the laboratory of Dr. Gus Lienhard (3).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the role of IRS-4 in IGF-I receptor signaling, we cloned hIRS-4 from HEK 293 cells, as described in Materials and Methods. The expression of functional IRS-4 was verified in NIH-3T3 mouse fibroblasts overexpressing the hIGF-I receptor (WTB3 cells) (8). WTB3 cells were transiently transfected with full-length (FL) hIRS-4. WTB3 cells contain little or no endogenous IRS-4, and display high levels of IGF-I receptor expression, thereby providing a good system to study the role of IRS-4 in IGF-I receptor signaling. Moreover, we have recently demonstrated that IRS-4 is involved in the mitogenic functions of the IGF-I receptor in WTB3 cells stably transfected with hIRS-4 (10). As shown in Fig. 1Go, when WTB3 cells were transiently transfected with the FL IRS-4 construct, stimulation with IGF-I resulted in tyrosine phosphorylation of both the IGF-I receptor and of IRS-4.



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  		Figure 1. IRS-4 expression in transiently transfected WTB3 cells. WTB3 cells were transfected with IRS-4-pCEFL KZ HA or mock transfected. Cells were incubated for 5 min in the presence or absence of 50 nM of IGF-I. IRS-4 was immunoprecipitated from whole cell lysates with an anti-IRS-4 Ab. Immunoprecipitated samples were then run on SDS-PAGE and blotted with either antiphosphotyrosine or anti-IRS-4 antibodies.

 
In a previous study, we have recently shown that upon phosphorylation, the IRS-4 molecule interacts with Crk proteins (6). We now sought to determine which part of the IRS-4 molecule is required for its interaction with the Crk-II protein. To address this issue, a series of N- and C-terminal truncation mutants were constructed as described in Materials and Methods. These mutants are shown in schematic form in Fig. 2AGo. The full-length IRS-4 and various mutants were transiently expressed in WTB3 cells. Cleared whole cell lysates from control and IGF-I stimulated cells were incubated with a recombinant Crk-II-SH2-SH3 GST fusion protein, and the interactions of this protein with full-length and various IRS-4 mutants were analyzed in GST pull-down experiments (Fig. 2BGo). IGF-I induced the interaction of Crk-II with full-length IRS-4 as well as with IRS-4 1–315 and IRS-4 1–678 N terminus truncated IRS-4. In contrast, the IRS-4 1–1140 protein failed to interact with Crk-II. We also found that the IRS-4 976-1257 and IRS-4 801-1257 C-terminus truncated IRS-4 mutant proteins retained their ability to interact with Crk-II, but that this interaction was abolished in the IRS-4 678-1257 mutant. These results indicate that the region of IRS-4 located between amino acids 678 and 800 is likely to be responsible for its IGF-I-induced interaction with Crk-II.

The region of IRS-4 located between amino acids 678 and 800 contains a cluster of tyrosines, including Y700, Y717, Y743, and Y779, which may be involved in the interaction of IRS-4 with the SH2 domain of Crk-II when IRS-4 becomes tyrosine phosphorylated by the IGF-I receptor. To test this hypothesis, we created a number of IRS-4 mutants, including Y700->F700, Y717->F717, Y743->F743, Y779->F779, the double mutants Y700Y717->F700F717 and Y743Y779->F743F779, and the quadruple mutant Y700Y717Y743Y779->F700F717F743F779. These mutants, along with wild-type IRS-4 proteins, were transiently expressed in WTB3 cells. Immunoprecipitation of the mutant and wild-type proteins with anti-IRS-4 antibodies followed by Western blotting demonstrated that expression levels of these proteins was approximately equal across the various transfections (Fig. 3AGo). Both wild-type and mutant IRS-4 proteins were tyrosine phosphorylated upon IGF-I receptor stimulation, as shown in Fig. 3A. Whole cell lysates from control and IGF-I-treated cells expressing wild-type or various mutants of IRS-4 were also analyzed in Crk-II-GST pulldown experiments. These experiments showed that IRS-4 interacts with Crk-II only when IRS-4 is tyrosine phosphorylated. In the quadruple tyrosine mutant (Y700Y717Y743Y779->F700F717F743F779, the interaction between IRS-4 and Crk-II was completely abolished (Fig. 3BGo). None of the single tyrosine mutations resulted in significant changes in the affinity of the Crk-II-GST protein for tyrosine phosphorylated IRS-4 (Fig. 3Go). However, there was a significant about 50% decrease in the interaction of the double mutants Y700Y717->F700F717 and Y743Y779->F743F779 with Crk-II.



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  		Figure 3. Mutational analysis of IRS-4 Crk-II interaction. WTB3 cells were transiently transfected with various constructs of hIRS-4, including wild-type (FL), Y700Y717Y743Y779->F700F717F743F779 (1 2 3 4 ), Y700Y717->F700F717 (1 2 ), Y743Y779->F743F779 (3 4 ), Y700->F700 (1 ), Y717->F717 (2 ), Y743->F743 (3 ), Y779->F779 lanes (4 ), or mock transfected (mock). WTB3 cells stably transfected with hIRS-4 (10 ) were included as a positive control (con). IRS-4 was immunoprecipitated from whole cell lysates with anti-hIRS-4 Ab (A) or lysates were used in Crk-II-GST pulldown experiments (B) as described in Materials and Methods. The resulting coassociated proteins were subjected to SDS-PAGE followed by immunoblotting with the anti-hIRS-4 Ab (upper panels in A and B). The blots were then stripped and reblotted with the anti-phosphotyrosine antibodies (lower panels in A and B). C, The results of three Crk-II-GST pulldown experiments were summarized. The results are presented as percent of pulldown of wild-type hIRS-4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Members of the IRS family of proteins are important intracellular substrates for the insulin and IGF-I receptor tyrosine kinase signaling cascades. All four IRS proteins contain highly conserved N-terminal PH and PTB domains followed by a C-terminal domain that contains numerous tyrosine phosphorylation motifs. The PH domains consist of approximately 100 amino acids with a conserved secondary structure that binds acidic motifs in proteins or phospholipids. While a direct interaction between the PH domain of the IRS molecules and the insulin or IGF-I receptors has not been demonstrated, its presence is essential for efficient tyrosine phosphorylation of IRS-1 by the insulin receptor. Deletion of the PH domain reduces insulin-stimulated IRS-1 tyrosine phosphorylation in the presence of normal levels of the insulin receptor (11). The PTB domain, on the other hand, interacts with the juxtamembrane regions of the insulin and IGF-I receptors via the conserved NPX(p)Y motif upon phosphorylation of this tyrosine residue by the receptor kinase (12, 13). This interaction of IRS molecules with the activated receptors leads to rapid phosphorylation of the IRS molecules on other tyrosine residues. These tyrosine residues lie in various motifs that provide binding sites for distinct SH2-containing proteins. For example, the regulatory subunit of PI3'kinase (p85) binds to YMXM and YXXM motifs in IRS-1 and IRS-2 (14), whereas Grb-2 binds YVNI (15) and the phosphatase SH-PTP2 binds YIDL and YASI motifs (15). Similar interactions have been observed with the newest members of the IRS family, IRS-3 and IRS-4. IRS-3, which is much smaller than the other IRS proteins, complexes strongly with p85, Nck, and SHP-2, and to a weaker extent with Grb-2 (16). The weak interaction with Grb-2 may be due to the absence of a specific motif in the shortened C-terminal region of IRS-3 compared with the other three IRS molecules. IRS-4 interacts strongly with p85 and Grb-2, but not with SHP-2 nor with phospholipase C{gamma} (17). Thus, there appears to be a degree of specificity were different IRS proteins show preferences for certain substrates.

Previous studies from our laboratory have identified another family of adapter proteins that are involved in IGF-I receptor signaling, and possibly with insulin receptor function (18, 19). The Crk family of proteins consists of v-Crk, Crk-I, Crk-II, and CrkL (20). v-Crk was originally discovered to be a viral oncogenic protein with an N-terminal gag sequence. Crk-I and Crk-II are cellular homologs of v-Crk that are splicing variants of one gene, with Crk-II being expressed ubiquitously and at higher levels than Crk-I. The CrkL protein is a separate gene product. All of these Crk proteins contain SH2 and SH3 domains with short intervening sequences, but lack a catalytic domain (20). Crk-II and CrkL have been shown to be involved in the signaling pathways of various tyrosine kinase receptors as well as in integrin signaling pathways (7). We have previously demonstrated that Crk-II interacts with three of the IRS molecules, IRS-1, IRS-2, and IRS-4 (6, 19). Following IGF-I stimulation of 293 human embryonic kidney cells, IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine residues as is Crk-II. Interestingly, this leads to a dissociation of the IRS-1/2-Crk complex (19). In contrast, Crk-II (and CrkL) show enhanced association with IRS-4 under these circumstances (6). These findings suggest that there is a degree of specificity determined by the various subtypes of IRS molecules and their interactions with particular receptors. We have also previously demonstrated that Crk-II and CrkL compete with each other for binding to IRS-4, suggesting that there is a common binding domain on the IRS-4 molecule. However, the specific domains of Crk-II and CrkL required for this interaction are different. The SH2 domain of Crk-II showed the maximum interaction with IRS-4, whereas both the SH2 and the N-terminal SH3 domain were required for maximal interaction of CrkL with IRS-4. The molecular basis of these differential interactions is likely to be due to the specific differences in the two molecules. Whereas Crk-II has a single phosphotyrosine residue (Tyr 221) lying between the two SH3 domains that binds its own SH2 domain, CrkL has additional phosphotyrosines that may allow for the formation of a different secondary structure following tyrosine phosphorylation. These differences may affect interactions with downstream molecules and may explain our findings that while CrkL is oncogenic, Crk-II is not and may lead to a differentiated phenotype.

In the present study, we set out to determine the exact region of the IRS-4 molecule that interacts with Crk-II. Our initial observations, using deletion constructs from both the N-terminal and C-terminal regions of IRS-4, suggested that Crk-II interacts with a region of the IRS-4 molecule that encompasses four potential tyrosine phosphorylation sites in motifs that predict interactions with SH2 domain-containing proteins. Indeed, the interaction of IRS-4 with Crk-II required IGF-I-induced tyrosine phosphorylation of IRS-4. The motifs surrounding tyrosine residues Y700, Y717, Y743, and Y779 include Y(p)VPM, Y(p)MPM, Y(p)MMM, and Y(p)MFM, respectively. Interestingly these four motifs are highly conserved between IRS-1, IRS-2, and IRS-4. Previous studies have suggested that Crk-II interacts with a specific Y(p)XXP motif (21). However, none of the phosphotyrosines in IRS-4 molecule are in such a motif. Because deletion analysis of proteins can remove important, though unidentified regions of the molecule, we chose instead to mutate all four tyrosines both individually and simultaneously to confirm their involvement in interactions with Crk-II. Indeed, simultaneous mutation of all four tyrosines totally abrogated the interaction of IRS-4 with Crk-II, confirming an important role of these residues in this interaction. To specifically identify which of the four tyrosine motifs in this cluster mediates the interaction of IRS-4 with Crk-II, we mutated each tyrosine separately and in various combinations. While individual mutations failed to reduced the interaction between IRS-4 and Crk-II, combinations of two mutant tyrosines had a strong effect, but these double mutation were nor as effective as was the quadruple mutant Y700Y717Y743Y779->F700F717F743F779. These results suggest that no single phosphotyrosine motif is involved in the IRS-4-Crk-II interaction. However, we cannot exclude the possibility that there is specificity for individual phosphotyrosine residues to interact with Crk-II that cannot be discerned from these studies. For example, it is possible that when individual tyrosine residues are mutated to phenylalanine, neighboring tyrosine residues can functionally substitute and serve as an alternative substrate for IGF-I receptor tyrosine kinase. The specific tyrosine residues within wild-type IRS-4 that are phosphorylated in vivo by the IGF-I receptor have not yet been established.

In summary, we have identified four specific tyrosine residues within the IRS-4 molecule as docking sites that are required for the interaction of IRS-4 with Crk-II following IGF-I receptor activation. These and future studies should advance our understanding of the role of the Crk family of proteins in mediating the functional effects of the IGF-I and insulin receptors.


   Footnotes
 
1 These authors equally contributed to this work. Back

2 Current address: Kimmel Cancer Institute and Department of Microbiology/Immunology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107. Back

Received November 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals