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Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes

1998; Springer Nature; Volume: 17; Issue: 9 Linguagem: Inglês

10.1093/emboj/17.9.2554

1460-2075

Shuichi Okada,

Metabolism, Diabetes, and Cancer

Article1 May 1998free access Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2–SOS and CrkII–C3G complexes Shuichi Okada Shuichi Okada Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author Michiyuki Matsuda Michiyuki Matsuda Department of Pathology, Research Institute, International Medical Center of Japan, Shinjuki-ku, Tokyo, 162 Japan Search for more papers by this author Mordechai Anafi Mordechai Anafi Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5 Search for more papers by this author Tony Pawson Tony Pawson Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5 Search for more papers by this author Jeffrey E. Pessin Jeffrey E. Pessin Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author Shuichi Okada Shuichi Okada Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author Michiyuki Matsuda Michiyuki Matsuda Department of Pathology, Research Institute, International Medical Center of Japan, Shinjuki-ku, Tokyo, 162 Japan Search for more papers by this author Mordechai Anafi Mordechai Anafi Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5 Search for more papers by this author Tony Pawson Tony Pawson Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5 Search for more papers by this author Jeffrey E. Pessin Jeffrey E. Pessin Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA, 52242 USA Search for more papers by this author Author Information Shuichi Okada1, Michiyuki Matsuda2, Mordechai Anafi3, Tony Pawson3 and Jeffrey E. Pessin1 1Department of Physiology and Biophysics, The University of Iowa, Iowa City, IA, 52242 USA 2Department of Pathology, Research Institute, International Medical Center of Japan, Shinjuki-ku, Tokyo, 162 Japan 3Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada, M5G 1X5 The EMBO Journal (1998)17:2554-2565https://doi.org/10.1093/emboj/17.9.2554 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1–Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII–C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII–C3G was recapitulated in vitro using a GST–C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST–C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade. Introduction The Rap proteins (Rap1A, Rap1B, Rap2A and Rap2B) are members of the Ras superfamily of low molecular weight monomeric GTP-binding proteins (Bokoch, 1993; Burgering and Bos, 1995). Rap1A was identified originally based upon its homology with the Drosophilia Ras-related gene (Dras3) and independently from its ability to induce a flat phenotype in v-Ki-Ras-transformed fibroblasts (Pizon et al., 1988; Kitayama et al., 1989). However, the physiological role of Rap1 in regulating intracellular signaling events appears to be enigmatic and can be either positive or negative dependent upon the particular cell context. Nevertheless, in multiple cell types, several studies have demonstrated that Rap1 can function as a suppressor of Ras-mediated downstream signaling. For example, both the Ras- and T-antigen-dependent transformation can be reversed by Rap1 expression (Kitayama et al., 1989; Jelink and Hassell, 1992). In addition, constitutively active Rap1 prevents Ras-induced germinal vesicle breakdown in Xenopus oocytes, and in mammalian cells antagonizes the Ras-dependent activation of the MAP kinase pathway, c–fos gene expression and the Ras-mediated inhibition of muscarinic potassium channel activity (Yatani et al., 1990; Campa et al., 1991; Sakoda et al., 1992; Cook et al., 1993). More recently, elevation of cAMP levels has been reported to activate Rap1 whereas, in several cell systems, cAMP functions to inhibit Ras downstream signals by blockade of the Raf/MEK/ERK activation pathway (Altshculer et al., 1995; Burgering and Bos, 1995). This apparent antagonism between Ras and Rap1 function may reflect the ability of Rap1 and Ras to interact with the same downstream effectors, since these proteins share identical sequences within their respective effector domains (Pizon et al., 1988; Kitayama et al., 1989; Zhang et al., 1990). In this regard, several studies have demonstrated that both Rap and Ras can bind the same regulators (p120RasGap) and effectors (RalGDS, Raf1 and B-Raf) in a GTP-dependent manner (Frech et al., 1990; Hata et al., 1990; Spaargaren and Bischoff, 1994; Nassar et al., 1995; Vossler et al., 1997). Over the past several years, substantial progress has been made in our understanding of the proximal molecular events controlling Ras activation and its coupling to downstream effector pathways. In the case of receptor tyrosine kinase activation, the tyrosine phosphorylation of the Shc proteins generates high affinity recognition motifs for the Src homology 2 (SH2) domain of Grb2 (Rozakis-Adcock et al., 1992; Medema and Bos, 1993; Downward, 1994; Chardin et al., 1995; van der Geer et al., 1996). Grb2 is a small adaptor protein (∼23 kDa) consisting of a single SH2 domain flanked by two Src homology 3 (SH3) domains (Lowenstein et al., 1992). In contrast to the SH2 domain which is responsible for its association with tyrosine-phosphorylated Shc, the Grb2 SH3 domains direct a basal state interaction with SOS, the 150 kDa guanylnucleotide exchange factor for Ras (Perrimon, 1994; Downward, 1996). Thus, receptor tyrosine kinase phosphorylation of Shc generates the formation of a Shc–Grb2–SOS ternary complex which provides a functional complex necessary for Ras activation. Once in the active GTP-bound state, Ras directly associates and activates the Raf1 kinase. In turn, activated Raf1 phosphorylates MEK which is an immediate upstream activator of ERK, a mitogen-activated protein (MAP) kinase (Avruch et al., 1994; Blumer and Johnson, 1994; Marshall, 1994). In an analogous paradigm, it is becoming clear that Rap1 activation (GTP-bound state) occurs through interaction with the Rap1-specific 140 kDa guanylnucleotide exchange factor C3G (Tanaka et al., 1994; Gotoh et al., 1995). In addition, increased expression of C3G can also suppress the v-Ki-Ras-transformed phenotype in a manner similar to increased expression of Rap1 (Gotoh et al., 1995). Furthermore, Crk represents another family of small adaptor proteins that are composed of SH2 and SH3 domains (Mayer et al., 1988; Matsuda et al., 1992; Reichman et al., 1992; ten Hoeve et al., 1994). In particular, CrkII is composed of a single N-terminal SH2 domain and two tandem SH3 domains. Under basal conditions, C3G is associated with CrkII through interactions of the central SH3 domain of CrkII with several proline-rich regions in C3G (Knudsen et al., 1994; Matsuda et al., 1994; Tanaka et al., 1994; Feller et al., 1995). Similarly to Grb2, the N-terminal SH2 domain can then direct the association of CrkII with several tyrosine-phosphorylated proteins including the epidermal growth factor (EGF) and nerve growth factor (NGF) receptors, IRS1/2, Cbl, paxillin and pp130Cas (Matsuda et al., 1990; Birge et al., 1992, 1993; Hempstead et al., 1994; Sakai et al., 1994; Teng et al., 1995; Beitner-Johnson et al., 1996; Ribon et al., 1996). Whether targeting of the CrkII–C3G complex to these tyrosine-phosphorylated proteins is responsible for the positive and/or negative signaling properties of Rap1 remains to be determined. In any case, since Rap1 can function as a suppressor of Ras downstream signaling, there must be a cellular mechanism that rapidly inhibits Rap1 function to allow for Ras activation of downstream signals. Recently, several studies have suggested that the activation of Ras can be limited by a negative feedback loop which results in the serine/threonine phosphorylation of SOS and dissociation of the Grb2–SOS complex (Cherniack et al., 1995; Langlois et al., 1995; Waters et al., 1995b; Holt et al., 1996a). We therefore hypothesized that a more proximal signaling pathway leading to the dissociation of CrkII from C3G may also exist to inactivate Rap1 prior to the activation of the Ras/Raf/MEK/ERK pathway. Here we demonstrate that insulin stimulation results in a rapid dissociation of the CrkII–C3G complex, inactivation of Rap1 and decreased association of Rap1 with the Raf1 serine kinase. In addition, the uncoupling of the CrkII–C3G complex occurs in a Ras-independent manner but is directly dependent upon the phosphorylation of CrkII at Tyr221. Results Insulin induces a time-dependent uncoupling of Raf1 from Rap1 with a concomitant association of Raf1 with Ras It has been well established that growth factor activation of Ras results in GTP-dependent association and activation of the Raf1 kinase (Moodie et al., 1993; Van Aelst et al., 1993; Vojtek et al., 1993; Warne et al., 1993; Zhang et al., 1993). In addition, Rap1 can also associate with Raf1 in a GTP-dependent manner (Ruggieri et al., 1994; Sprang, 1995; Herrmann et al., 1996; Hu et al., 1997; Vossler et al., 1997). To examine the reciprocal relationship between Raf1 and these two GTP-binding proteins, we immunoprecipitated Raf1 from control and insulin-stimulated cells and compared the relative co-immunoprecipitation of Rap1 and Ras (Figure 1). In the absence of insulin, Rap1 was co-immunoprecipitated specifically with Raf1 (Figure 1A, lane 1). However, following various times of insulin stimulation, there was a time-dependent decrease in the amount of Raf1 associated with Rap1 (Figure 1A, lanes 1–6). Following 1 min of insulin stimulation, there was a detectable decrease in the amount of Raf1-associated Rap1 protein (Figure 1A, lanes 1 and 2). The maximal decrease in the amount of Rap1 co-immunoprecipitated with Raf1 occurred by 5 min and remained at reduced levels from 15 to 30 min (Figure 1A, lanes 5 and 6). These differences were not due to changes in Raf1 immunoprecipitation, as Raf1 immunoblots of these precipitates demonstrate identical amounts of Raf1 protein (Figure 1A, lanes 7–12). Figure 1.Insulin stimulation decreases the amount of Raf1 associated with Rap1 concomitant with an increase in the amount of Raf1 associated with Ras. CHO/IR cells were incubated in the absence (lanes 1 and 7) or presence of 100 nM insulin for 1 (lanes 2 and 8), 3 (lanes 3 and 9), 5 (lanes 4 and 10), 15 (lanes 5 and 11) and 30 min (lanes 6 and 12) at 37°C. Whole cell detergent extracts were prepared and were immunoprecipitated with a Raf1 antibody as described in Materials and methods. (A) The Raf1 immunoprecipitates were then immunoblotted with a Rap1 antibody (lanes 1–6) or a Raf1 antibody (lanes 7–12). (B) The Raf1 immunoprecipitates were also immunoblotted with a Ras antibody (lanes 1–6) or a Raf1 antibody (lanes 7–12). Download figure Download PowerPoint Concomitant with the insulin-stimulated decrease in association between Rap1 and Raf1, there was a reciprocal increase in the amount of Raf1 that was associated with Ras (Figure 1B). Insulin stimulation for 1–3 min resulted in an increased amount of Ras co-immunoprecipitated with Raf1 compared with the unstimulated cells (Figure 1B, lanes 1–3). However, at longer times, there was a subsequent decline back towards basal levels (Figure 1B, lanes 4–6). Following 30 min of insulin stimulation, the amount of Ras associated with Raf1 was similar to that observed in the unstimulated cells. As a control for immunoprecipitation, the amount of Raf1 immunoprecipitated at all these time points was essentially constant, as determined by Raf1 immunoblotting of the Raf1 immunoprecipitates (Figure 1B, lanes 7–12). Insulin stimulation results in a rapid inactivation of Rap1 Insulin has been well documented to stimulate Ras activation through the regulated exchange of GDP for GTP by the guanylnucleotide exchange factor SOS (Maassen et al., 1992; Kahn, 1994; Roth et al., 1994). To examine the GTP-binding status of Rap1, we utilized the Rap1-binding domain (RBD) of RalGDS to precipitate GTP-bound Rap1 as recently described by Franke et al. (1997). In the absence of insulin, GST–RBD precipitated Rap1 from whole cell detergent extracts (Figure 2, lane 1). However, insulin stimulation resulted in a time-dependent decrease in the amount of Rap1 that was precipitated with GST–RBD (Figure 2, lanes 2–5). There was a detectable decrease in the amount of Rap1 precipitated by GST–RBD following 1 min of insulin stimulation, and this was maximally reduced by 5 min. This was not a result of differences in the amount of Rap1 present in the cell extracts as determined by direct immunoblotting (Figure 2, lanes 6–10). Thus, in contrast to Ras in which insulin stimulation results in rapid activation, Rap1 is apparently converted to the inactive GDP-bound state. Figure 2.Insulin stimulation results in a time-dependent inactivation of Rap1. CHO/IR cells were incubated in the absence (lanes 1 and 6) or presence of 100 nM insulin for 1 (lanes 2 and 7), 3 (lanes 3 and 8), 5 (lanes 4 and 9) and 15 min (lanes 5 and 10) at 37°C. Whole cell detergent extracts were prepared and incubated in the presence (lanes 1–5) or absence (lanes 6–10) of the GST–RBD fusion proteins as described in Materials and methods. The samples were then immunoblotted with a Rap1-specific antibody. Download figure Download PowerPoint Expression of Rap1 reduces insulin-stimulated Ras–Raf interaction If insulin induces an inactivation of endogenous Rap1, then overexpression of Rap1 would be expected to saturate the system and thereby prevent this inactivation pathway. Therefore, to determine if Rap1 can antagonize Ras function, we expressed the wild-type Rap1 and determined the extent of insulin-stimulated Ras–Raf1 association (Figure 3). In empty vector-transfected cells, insulin stimulation for 1 min resulted in an increased amount of Ras co-immunoprecipitated with Raf1 (Figure 3A, lanes 1 and 2). Similarly, insulin stimulation for 5 min also resulted in a greater extent of Ras co-immunoprecipitated with Raf1 compared with unstimulated cells, but this was somewhat less than that observed at 1 min (Figure 3A, lane 3). In contrast, expression of Rap1 resulted in a marked diminution in the amount of Ras that was co-immunoprecipitated with Raf1 in unstimulated cells (Figure 3A, lane 4). In addition, the ability of insulin to increase the extent of Ras–Raf1 association was greatly attenuated (Figure 3A, lanes 5 and 6). To ensure equal immunoprecipitation of Raf1 under these conditions, the Raf1 immunoprecipitates were also immunoblotted for Raf1 (Figure 3B, lanes 1–6). Similarly, there was no significant change in the total cellular expression of Raf1 following Rap1 transfection as determined by Raf1 immunoblots of the same whole cell extracts (data not shown). Interestingly, although insulin had no effect on the amount of Ras protein present in the cell extracts, expression of Rap1 resulted in a small compensatory increase in the cellular content of Ras (Figure 3C, lanes 1–6). Nevertheless, the inhibition in Ras–Raf1 interaction directly correlated with a reduction in insulin-stimulated ERK activation (data not shown). The Rap1 inhibition of ERK activation occurred to the same extent and with the same time frame as previously reported (Cook et al., 1993). Thus, these data demonstrate that in this cell system Rap1 functions as a competitor for the association of Raf1 with Ras. Figure 3.Increased expression of Rap1 reduces the amount of Raf1 associated with Ras. CHO/IR cells were transfected by electroporation with 100 μg of the empty vector (Vector, lanes 1–3) or with the same vector coding for Rap1 (Rap1, lanes 4–6). At 48 h following transfection, the cells were either left untreated (lanes 1 and 4) or stimulated with 100 nM insulin for 1 (lanes 2 and 5) and 5 min (lanes 3 and 6) at 37°C. Whole cell detergent extracts were prepared, immunoprecipitated with a Raf1 antibody and immunoblotted for Ras (A) or Raf1 (B) as described in Materials and methods. (C) The whole cell extracts (lysates) were also immunoblotted directly for Ras. Download figure Download PowerPoint Insulin stimulation results in the dissociation of CrkII from C3G which precedes the dissociation of the Grb2–SOS complex We and others recently have observed that the inactivation of Ras back to the GDP-bound state occurs concomitant with the dissociation of the Grb2–SOS complex (Cherniack et al., 1995; Langlois et al., 1995; Waters et al., 1995a). We therefore speculated that the decrease in Rap1 GTP binding might have resulted from the inactivation of Rap1 due to a rapid uncoupling of the CrkII–C3G complex. Thus, to address the mechanism by which insulin induces an inactivation of Rap1, we compared the relationship between Grb2–SOS and CrkII–C3G interactions by co-immunoprecipitation (Figure 4). As expected, in unstimulated cells, immunoprecipitation of C3G resulted in the co-immunoprecipitation of CrkII (Figure 4A, lane 1). However, following insulin stimulation, there was a time-dependent decrease in the amount of CrkII that could be co-immunoprecipitated with C3G (Figure 4A, lanes 1–6). The dissociation of CrkII from C3G was detectable as early as 1 min, and continued to decline over the 30 min time period examined. The insulin-stimulated decrease of C3G-immunoprecipitated CrkII protein was not due to differences in C3G immunoprecipitation as assessed by C3G immunoblotting of the C3G immunoprecipitates (Figure 4A, lanes 7–12). In addition, there was no change in the total amount of CrkII in the whole cell detergent extracts (data not shown). Figure 4.Insulin stimulation results in the dissociation of the CrkII–C3G complex. CHO/IR cells were incubated in the absence (lanes 1 and 7) or presence of 100 nM insulin for 1 (lanes 2 and 8), 3 (lanes 3 and 9), 5 (lanes 4 and 10), 15 (lanes 5 and 11) and 30 min (lanes 6 and 12) at 37°C. Whole cell detergent extracts were prepared and were immunoprecipitated with a C3G antibody (A) or a SOS antibody (B) as described in Materials and methods. The C3G immunoprecipitates (upper panel) were immunoblotted with a CrkII antibody (lanes 1–6) or a C3G antibody (lanes 7–12). The SOS immunoprecipitates (lower panel) were immunoblotted with a Grb2 antibody (lanes 1–6) or a SOS antibody (lanes 7–12). Download figure Download PowerPoint In comparison with the CrkII–C3G complex, insulin stimulation also resulted a time-dependent dissociation of the Grb2–SOS complex (Figure 4B, lanes 1–6). However, as previously reported (Waters et al., 1995b), the ability of insulin to induce the dissociation of Grb2–SOS was not detectable until ∼5 min and was maximal by ∼15 min (Figure 4B, lanes 4 and 5). As controls, the amount of SOS protein immunoprecipitated under these conditions was essentially identical (Figure 4B, lanes 7–12). Together, these data demonstrate that insulin stimulation results in a rapid dissociation of the CrkII–C3G complex which precedes the dissociation of the Grb2–SOS complex. The insulin-stimulated dissociation of the CrkII–C3G complex is Ras independent The dissociation of the Grb2–SOS complex results from a serine/threonine phosphorylation of SOS, which can be prevented by inhibition of Ras function or by inhibiting the downstream kinase MEK (Langlois et al., 1995; Waters et al., 1995a,b). As observed in Figure 4, insulin stimulation resulted in the dissociation of the Grb2–SOS complex as assessed by SOS immunoprecipitation followed by Grb2 immunoblotting (Figure 5A, lanes 1 and 2). In contrast, expression of the dominant-interfering Ras mutant (N17Ras) completely prevented the insulin-stimulated dissociation of Grb2 from SOS (Figure 5A, lanes 3 and 4). However, in the same cell extracts, the ability of insulin to dissociate the CrkII–C3G complex occurred in both the absence and presence of N17Ras (Figure 5B, lanes 1–4). Thus, unlike the insulin regulation of the Grb2–SOS complex, the insulin-stimulated dissociation of the CrkII–C3G complex occurred in a Ras-independent manner. Figure 5.The insulin-stimulated dissociation of the CrkII–C3G complex occurs through a Ras-independent mechanism. CHO/IR cells were quantitatively electroporated with an empty vector or with the vector encoding the dominant-interfering Ras mutant, N17Ras, as described in Materials and methods. The cells either were left untreated or stimulated with 100 nM insulin for 20 min at 37°C followed by preparation of whole cell detergent extracts. The extracts were then (A) immunoprecipitated with a SOS antibody and immunoblotted for Grb2 or (B) immunoprecipitated with a C3G antibody and immunoblotted for CrkII. Download figure Download PowerPoint Dissociation of the CrkII–C3G complex results from a decreased affinity of CrkII for C3G There are several possible mechanisms that could account for the insulin-stimulated dissociation of the CrkII–C3G complex. The most direct, and therefore the most likely, would be a rapid functional alteration of CrkII and/or C3G. To determine whether the reduced association of CrkII and C3G was due a modification in either of these two proteins, we examined the ability of GST–CrkII and GST–C3G fusion proteins to precipitate their corresponding binding partner (Figure 6). Incubation of detergent cell extracts isolated from control cells with 4 or 8 μg of glutathione–Sepharose demonstrated no specific binding to C3G, as determined by immunoblotting with a C3G antibody (Figure 6A, lanes 1 and 3). Similarly, there was no specific C3G binding in GST precipitates of detergent cell extracts isolated from insulin-stimulated cells (Figure 6A, lanes 2 and 4). In contrast, incubation of the detergent cell extracts with either 4 or 8 μg of GST–CrkII resulted in the precipitation of C3G (Figure 6A, lanes 5–8). Under these conditions, there was no significant difference in the amount of C3G precipitated from control and insulin-stimulated extracts (Figure 6A, compare lane 5 with 6, and lane 7 with 8). Figure 6.The insulin-stimulated dissociation of the CrkII–C3G complex can be recapitulated in vitro. CHO/IR cells were incubated in the absence (lanes 1, 3, 5 and 7) or presence (lanes 2, 4, 6 and 8) of 100 nM insulin for 5 min at 37°C. Whole cell detergent extracts were prepared and the samples were then incubated for 1 h at 4°C with either 4 (lanes 1, 2, 5 and 6) or 8 μg (lanes 3, 4, 7 and 8) of GST, GST–CrkII and GST–C3Gpro fusion proteins. The resultant precipitates were immunoblotted with either a C3G antibody (A) or a CrkII antibody (B). Download figure Download PowerPoint Similarly, incubation of the detergent cell extracts with glutathione–Sepharose demonstrated no specific association with CrkII (Figure 6B, lanes 1–4). Due to the relatively large molecular weight of C3G which precluded the preparation of a full-length GST–C3G fusion protein, we prepared a truncated GST fusion protein containing the proline-rich domain of C3G (GST–C3Gpro). This domain has been shown previously to interact specifically with the middle SH3 domain of CrkII (Matsuda et al., 1994). As expected, incubation of the cell extracts with GST–C3Gpro demonstrated a dose-dependent association of CrkII (Figure 6B, lanes 5–8). However, the amount of CrkII that was precipitated with GST–C3Gpro was significantly less from insulin-stimulated cell extracts compared with control cell extracts (Figure 6B, compare lane 5 with 6, and lane 7 with 8). The insulin-stimulated reduction in CrkII association was detected at both concentrations of GST–C3Gpro used, but appeared to be less at 8 μg of GST–C3Gpro compared with 4 μg. Nevertheless, there was no significant difference in the total amount of either C3G or CrkII in the detergent cell extracts isolated from control or insulin-stimulated cells (data not shown). Since these data suggested that the reduction in CrkII association with GST–C3Gpro was concentration dependent, we next examined the binding of CrkII to various amounts of GST–C3Gpro (Figure 7). CrkII immunoblots of whole cell detergent extracts demonstrated the presence of the two typical bands migrating at ∼40 and 42 kDa, respectively (Figure 7A, lane 1). Insulin stimulation resulted in a relative increase in the amount of the 42 kDa band concomitant with a reduction in the 40 kDa band (Figure 7A, lane 2). This change in the proportion of the 40 and 42 kDa CrkII proteins was due to insulin-stimulated phosphorylation which resulted in a decreased mobility of the 40 kDa band (see below). As observed in Figure 5, precipitation with 4 and 8 μg of GST–C3Gpro resulted in the appearence of only the 40 kDa band (Figure 7A, lanes 3–6). Again, there was a reduction in the amount of CrkII that was precipitated from the insulin-stimulated cell extracts compared with the control extracts (Figure 7A, compare lane 3 with 4, and lane 5 with 6). However, with increasing concentrations of GST–C3Gpro (32 and 120 μg), the difference between the control and insulin-stimulated cell extracts was no longer detectable (Figure 7A, compare lane 7 with 8, and lane 9 with 10). The saturation of CrkII binding was also specific at these concentrations of GST–C3Gpro as there was no detectable precipitation of CrkII by GST alone (data not shown). In addition, at these higher concentrations of GST–C3Gpro, the presence of the slower migrating 42 kDa CrkII protein was readily apparent, with no significant effect of insulin on its relative in vitro binding properties. Quantitation of several in vitro binding experiments by phosphorimager analysis demonstrated that insulin induced an ∼2.5-fold reduction in binding affinity, with no change in the number of binding sites (data not shown). Figure 7.The reduction of CrkII–C3G binding in vitro is due to a decrease in CrkII binding affinity. CHO/IR cells were incubated in the absence (lanes 1, 3, 5, 7 and 9) or presence (lanes 2, 4, 6, 8 and 10) of 100 nM insulin for 5 min at 37°C. Whole cell detergent extracts were prepared and the samples (3 mg) were then incubated for 1 h at 4°C with 4 (lanes 3 and 4), 8 (lanes 5 and 6), 32 (lanes 7 and 8) and 120 μg (lanes 9 and 10) of the GST–C3Gpro fusion protein. The original cell extracts (lysate lanes 1 and 2; 20 μg) as well as the resultant precipitates (lanes 3–10) were immunoblotted with a CrkII antibody. Download figure Download PowerPoint In an analogous manner, incubation of a fixed amount of GST–C3Gpro with varying concentrations of detergent cell extracts also demonstrated a dose-dependent binding of CrkII (data not shown). Thus, these data demonstrate that insulin stimulation results in a modification of CrkII that reduces its ability to associate with C3G in vitro. Furthermore, the decreased binding between CrkII and C3G occurred due to a change in binding affinity, with no significant alteration in the number of binding sites and, hence, expression levels of either the CrkII or C3G proteins. Insulin stimulates the tyrosine phosphorylation of CrkII In contrast to most small SH2/SH3 adaptor proteins, CrkII undergoes tyrosine phosphorylation by the c-Abl, NGF and insulin-like growth factor 1 (IGF1) receptor tyrosine kinases (Feller et al., 1994; ten Hoeve et al., 1994; Beitner-Johnson and LeRoith, 1995; Ribon and Saltiel, 1996). To determine whether insulin stimulation also resulted in the tyrosine phosphorylation of CrkII, CrkII immunoprecipitates were immunoblotted with the PY20 phosphotyrosine antibody (Figure 8). In unstimulated cells, there was a basal level of tyrosine-phosphorylated CrkII which was increased following 1 min of insulin stimulation (Figure 8A, lanes 1 and 2). The insulin-stimulated CrkII tyrosine phosphorylation was also rapid in that the maximal increase occurred within 1 min and remained constant for up to 30 min (Figure 8A, lanes 3–6). Figure 8.Insulin stimulation increases the extent of tyrosine-phosphorylated CrkII. CHO/IR cells were incubated in the absence (lane 1) or presence of 100 nM insulin for 1 (lane 2), 3 (lane 3), 5 (lane 4), 15 (lane 5) and 30 min