Growth Hormone Stimulates the Formation of a Multiprotein Signaling Complex Involving p130 Cas and CrkII

We have demonstrated previously that growth hormone (GH) activates focal adhesion kinase (FAK), and this activation results in the tyrosine phosphorylation of two FAK substrates, namely paxillin and tensin. We now show here in Chinese hamster ovary cells stably transfected with rat GH receptor cDNA that human (h)GH induces the formation of a large multiprotein signaling complex centered around another FAK-associated protein, p130Cas and the adaptor protein CrkII. hGH stimulates the tyrosine phosphorylation of both p130Cas and CrkII, their association, and the association of multiple other tyrosine-phosphorylated proteins to the complex. Both the c-Src and c-Fyn tyrosine kinases are tyrosine phosphorylated and activated by cellular hGH stimulation and form part of the multiprotein signaling complex as does tensin, paxillin, IRS-1, the p85 subunit of phosphatidylinositol 3-kinase, C3G, SHC, Grb-2, and Sos-1. c-Cbl and Nck are also tyrosine-phosphorylated by cellular stimulation with hGH and associate with the p130Cas-CrkII complex. c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is activated in response to hGH in accordance with the formation of the abovementioned signaling complex, and hGH stimulated JNK/SAPK activity is increased in CrkII overexpressing NIH3T3 cells compared with vector transfected NIH3T3 cells. The formation of such a large multiprotein signaling complex by GH, with the resultant activation of multiple downstream effector molecules, may be central to many of the pleiotropic effects of GH.

Growth hormone (GH) 1 is the major regulator of postnatal body growth (1). It possesses diverse and pleiotropic effects on the growth, differentiation, and metabolism of cells. GH is thought to initiate its biological actions, including induction of a number of RNA species in mammalian tissues by interaction with a specific membrane-bound receptor (2). The GH receptor is phosphorylated upon ligand stimulation presumably by the physical association of the nonreceptor tyrosine kinase JAK2 (3). The JAK kinases are linked to transcriptional regulation and JAK activation results in the phosphorylation, dimerization, and nuclear translocation of latent cytoplasmic STAT transcription factors (4). The activated STAT factors bind to their appropriate DNA-responsive elements to activate gene transcription (4). Other diverse actions of GH include the stimulation of chemotaxis and migration of monocytic cells (5). Concordantly, we have demonstrated that GH stimulates the re-organization of the actin cytoskeleton in cells with fibroblastic morphology (6) and reported that this re-organization of the actin cytoskeleton requires the activity of PI 3-kinase.
Focal adhesion kinase (p125 FAK ) has been postulated to play a central role in the response of the cell to the extracellular matrix (for review, see Ref. 7) and in cell morphology and motility (for review, see Ref. 8). For example, overexpression of FAK stimulates cell migration (9) and both FAK-deficient endodermal and mesodermal cells migrate slower than their FAK replete counterparts (10,11). The effect of FAK on the cytoskeleton is presumably mediated by the formation of a p130 Cas (Crk-associated substrate)-CrkII complex (12) in association with c-Src (for review, see Ref. 13). We have reported recently that GH stimulates the tyrosine phosphorylation of FAK and two of its substrates, paxillin and tensin, and that FAK associates with JAK2 and requires the JAK2 binding site on the GH receptor for its phosphorylation (14). We demonstrate here that hGH also stimulates the tyrosine phosphorylation of both p130 Cas and CrkII, their association, and the association of multiple other tyrosine-phosphorylated proteins to the complex including the c-Src and c-Fyn protein-tyrosine kinases, Nck, c-Cbl, tensin, paxillin, IRS-1, the p85 subunit of PI 3-kinase, SHC, Grb-2, Sos-1, and C3G. Finally we demonstrate that JNK/SAPK is activated in response to hGH in accordance with the formation of the p130 Cas -CrkII complex (15). The formation of such a large multiprotein signaling complex, with the potential downstream activation of multiple signaling pathways, may be central to many of the pleiotropic effects of GH, including cytoskeletal re-organization, cell migration, chemotaxis, mitogenesis, and/or prevention of apoptosis and gene transcription. ethanol. Blots were then washed for 30 min with several changes of phosphate-buffered saline, 0.1% Tween 20 at room temperature. Efficacy of stripping was determined by re-exposure of the membranes to ECL. Thereafter, blots were reblocked and immunolabeled as described above. Densitometry was performed by using the GS-700 imaging densitometer from Bio-Rad according to the manufacturer's instructions.
Kinase Assays-Src and Fyn kinase assays were performed as described according to the manufacturer's instructions (Upstate Biotechnology Inc.). In brief, supernatant containing 150 g of protein per sample derived from cells stimulated with hGH was incubated with 1 g of Src polyclonal antibody or 1 g of Fyn polyclonal antibody at 4°C overnight in a final volume of 500 l. Immunocomplexes were collected by incubation with 20 l of protein A/G plus-agarose for 1 h. Immunoprecipitates were washed three times with ice-cold lysis buffer. 10 l (150 M final concentration) of the substrate peptide, 10 l of Src reaction buffer, and 10 l of [␥-32 P]ATP stock were added to a microcentrifuge tube and incubated for 10 min at 30°C with agitation. 20 l of 40% trichloroacetic acid was then added to precipitate peptides, and a 25-l aliquot was transferred onto the center of a numbered P81 paper square. The assay squares were washed three times for 5 min each with 0.75% phosphoric acid and once with acetone. The assay squares were transferred to a scintillation vial, 5 ml of scintillation mixture added, and the level of radioactivity determined in a scintillation counter. JNK/SAPK assays were performed according to the manufacturer's instructions. In brief, CHO-GHR 1-638 cells were serum-deprived for 16 h, treated with 50 nM hGH, and the cells lysed at 4°C in 1 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerol phosphate, 1 mM Na 3 VO 4 , 0.1% phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin) per sample. The lysates were centrifuged at 15,000 ϫ g for 15 min at 4°C. The supernatant containing 200 g of protein per sample was incubated overnight at 4°C with 2 g of N-terminal c-Jun (1-89) fusion protein bound to glutathione-Sepharose beads in a final volume of 500 l in 1 ϫ lysis buffer. The beads were washed twice with 500 l of lysis buffer containing 0.1% phenylmethylsulfonyl fluoride and twice with 500 l of kinase buffer (25 mM Tris, pH 7.5, 5 mM glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ). The kinase reactions were carried out in the presence of 100 M ATP at 30°C for 30 min. c-Jun phosphorylation was selectively detected by Western immunoblotting using a chemiluminescent detection system and specific c-Jun antibodies (1:1000 dilution) recognizing phosphorylation of c-Jun at serine 63.
Statistics and Presentation of Data-All experiments were repeated at least three times, usually five to seven times. Figures presented for Western blot analyses are representative of multiple experiments. The text under "Results" summarizes the results from multiple Western blot analyses. Consequently, the text in the results section (e.g. description of the time course of phosphorylation and dephosphorylation) may not exactly correspond to the actual figure presented. All numerical data are expressed as mean Ϯ S.D. Data were analyzed using the two-tailed t test or analysis of variance.

RESULTS
hGH Stimulates the Tyrosine Phosphorylation of p130 Cas -We have reported previously that hGH stimulation of CHO-GHR 1-638 cells results in the tyrosine phosphorylation of FAK and two of its substrates, namely paxillin and tensin (14). Another protein associated with FAK is p130 Cas , although it is apparent that p130 Cas is tyrosine-phosphorylated by Src protein tyrosine kinase family members associated with FAK (19,20). To determine whether hGH could also stimulate the tyrosine phosphorylation of p130 Cas , we first treated CHO-GHR 1-638 cells for 0 -60 min with 50 nM hGH, prepared cell extracts, and immunoprecipitated tyrosine-phosphorylated proteins with PY20. SDS-PAGE and subsequent Western blotting for p130 Cas revealed a hGH-and time-dependent increase in the level of p130 Cas protein in the phosphotyrosine immunoprecipitates (Fig. 1A). p130 Cas protein was identified as a single band of 130 kDa. Some basal level of p130 Cas tyrosine phosphorylation was observed in serum-deprived cells. hGH-stimulated tyrosine phosphorylation of p130 Cas was first observed 2 min after stimulation. The maximal hGH-stimulated tyrosine phosphorylation of p130 Cas was observed between 5 and 30 min after stimulation, with the relative level of p130 Cas tyrosine phosphorylation decreasing at 60 min. We next immunoprecipitated p130 Cas from cell extracts of CHO-GHR 1-638 cells and examined the level of tyrosine phosphorylation by Western blot analysis with PY20. We observed the consistent presence of a time-and hGH-dependent tyrosine-phosphorylated band of 130 kDa, which co-migrated with p130 Cas protein ( Fig. 2A). Increased hGH-dependent tyrosine phosphorylation of p130 was observed first at 2 min, persisted to 30 min, and then declined at 60 min. p130 Cas has been reported to associate with FAK in a Src-dependent manner (for review, see Ref. 13). Other hGHdependent tyrosine-phosphorylated proteins were also observed in the p130 Cas immunoprecipitates and will be discussed below. However, one of these hGH-dependent tyrosinephosphorylated proteins possessed a molecular mass of 125 kDa. We therefore proceeded to determine the effect of cellular stimulation with hGH on the association state between p130 Cas and p125 FAK . Immunoprecipitation of p130 Cas from cell extracts and Western blot analysis for FAK revealed a time-and hGH-dependent association of FAK with p130 Cas (Fig. 2C). Constitutive association between FAK and p130 Cas was observed, and hGH stimulation of cells resulted in increased p130 Cas -FAK association observed first at 2 min, maximal between 5-30 min, and then followed by a decline to 60 min. Thus hGH stimulated the formation of a FAK-p130 Cas complex. p130 Cas has also been reported to associate with the adaptor protein CrkII (19). Immunoprecipitation of CrkII from cell extracts and subsequent Western blot analysis for p130 Cas revealed a time-and hGH-dependent association of p130 Cas with CrkII (Fig. 2E). hGH stimulation of cells resulted in increased p130 Cas -CrkII association between 2 and 30 min and followed by a decline at 60 min.
hGH Stimulates the Tyrosine Phosphorylation of CrkII and Formation of a Signaling Complex-To determine whether hGH could also stimulate the tyrosine phosphorylation of . CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and p130 Cas protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for tyrosine phosphorylation with the use of PY20. The loading control for p130 Cas is shown in B. The arrowhead marks the position of the tyrosine-phosphorylated band detected at 130 kDa. C, time dependence of the hGH-stimulated association of FAK with p130 Cas in CHO cells stably transfected with GH receptor cDNA (CHO-GHR 1-638 ). CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and p130 Cas protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for FAK. The loading control for p130 Cas is shown in D. The arrowhead marks the position of p125 FAK detected at 125 kDa. E, time dependence of the hGH-stimulated association of p130 Cas with CrkII in CHO cells stably transfected with GH receptor cDNA (CHO-GHR 1-638 ). CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and CrkII protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for p130 Cas . The loading control for CrkII is shown in F. The arrowhead marks the position of p130 Cas detected at 130 kDa. The position of the molecular mass standards is indicated on the right-hand side. The data presented are representative of at least three separate experiments. CrkII, we examined the level of CrkII protein in the phosphotyrosine (PY20) immunoprecipitates from extracts of cells treated with hGH (Fig. 1B). CrkII protein was identified as a doublet band of 40 and 42 kDa concordant with previous reports demonstrating the existence of 40-and 42-kDa CrkII (22); the 42-kDa CrkII being a phosphorylated form of CrkII with subsequent slower electrophoretic migration (23). Some basal level of CrkII tyrosine phosphorylation was observed in serum deprived cells. hGH-stimulated tyrosine phosphorylation of CrkII was first observed 2 min after stimulation. The 42-kDa CrkII was the major tyrosine-phosphorylated CrkII species following hGH stimulation. The maximal hGH-stimulated tyrosine phosphorylation of CrkII was observed between 2 and 30 min after stimulation, with the relative level of CrkII tyrosine phosphorylation decreasing at 60 min. From the above results it is apparent that CrkII is a component of a larger signaling complex. We therefore immunoprecipitated CrkII from cell extracts of CHO-GHR 1-638 cells stimulated for 0 -60 min with 50 nM hGH and examined the level of tyrosine phosphorylation of CrkII and CrkII-associated proteins by Western blot analysis with PY20 (Fig. 3A). We observed the consistent presence of major time-and hGH-dependent tyrosine-phosphorylated bands of 130, 125, 120, 85 (not obvious on blot presented), 68, 62, 60, 47, and 42 kDa. The tyrosine-phosphorylated band at 42 kDa co-migrated with the 42-kDa CrkII protein. The other hGH-dependent tyrosine-phosphorylated proteins associated with CrkII have been identified below. Other tyrosine-phosphorylated proteins were obscured by the presence of the IgG heavy chains. Also, when conditions of separation and transfer were optimized, larger molecular mass proteins at 180 and 215 kDa associated with CrkII were also observed to be tyrosinephosphorylated in response to cellular stimulation with hGH (identified below as IRS-1 and tensin, respectively). Increased hGH-dependent tyrosine phosphorylation of CrkII and CrkIIassociated proteins was generally observed first at 2 min, persisted to 30 min, and then declined at 60 min. We also proceeded to determine the effect of cellular stimulation with hGH on the association state between CrkII and FAK. Immunoprecipitation of CrkII from cell extracts and Western blot analysis for FAK revealed a time-and hGH-dependent association of FAK with CrkII ( Fig. 3C). hGH stimulation of cells resulted in increased FAK-CrkII association observed first at 2 min, maximal between 2 and 30 min, and then followed by a decline at 60 min. We also performed the reciprocal experiment with immunoprecipitation of FAK protein and immunoblotting for the presence of CrkII protein. This experiment depicted in Fig. 3E also similarly demonstrated the hGH dependence of the FAK-CrkII association. Thus hGH stimulated the formation of a FAK-p130 Cas -CrkII complex of signal-transducing proteins.
hGH Stimulates c-Src Kinase Activity-c-Src has been reported to associate with FAK and is responsible for full tyrosine phosphorylation of the FAK molecule as well as serving as an adaptor molecule for the association of FAK and p130Cas (for review, see Ref. 13). We also observed above the presence of a hGH-dependent tyrosine-phosphorylated protein of 60 kDa associated with the CrkII-p130 Cas complex. To determine whether hGH could also stimulate the tyrosine phosphorylation of c-Src, we immunoprecipitated tyrosine-phosphorylated proteins from cell extracts with PY20 and observed by Western blot analysis for c-Src, a hGH-and time-dependent increase in the level of c-Src protein in the phosphotyrosine immunoprecipitates (Fig. 1C). c-Src protein was identified as a single band of 60 kDa. c-Src was basally tyrosine-phosphorylated in serumdeprived cells. hGH-stimulated tyrosine phosphorylation of c-Src was first observed 2 min after stimulation. The maximal hGH-stimulated tyrosine phosphorylation of c-Src was ob-served between 2 and 15 min after stimulation, with the relative level of c-Src tyrosine phosphorylation decreasing at 60 min. We also performed the reciprocal experiment by immunoprecipitation of c-Src and Western blot analysis with PY20 and observed a major time-and hGH-dependent tyrosine-phosphorylated band of 60 kDa, which co-migrated with c-Src protein (Fig. 4A). Increased hGH-dependent tyrosine phosphorylation of c-Src was also observed first at 2 min, persisted to 15 min, and then declined at 30 and 60 min. We next proceeded to determine the association state between c-Src and FAK and the p130 Cas -CrkII complex. We demonstrated a time-and hGH-de-FIG. 3. A, time dependence of the hGH-stimulated tyrosine phosphorylation of CrkII and CrkII-associated proteins in CHO cells stably transfected with GH receptor cDNA (CHO-GHR 1-638 ). CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and CrkII protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for tyrosine phosphorylation with the use of PY20. The arrowheads mark the position of the tyrosine-phosphorylated bands detected and the IgG heavy chain. C, time dependence of the hGH-stimulated association of CrkII with FAK in CHO cells stably transfected with GH receptor cDNA (CHO-GHR 1-638 ). CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and FAK protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for CrkII. The loading control for FAK is shown in D. The arrowhead marks the position of CrkII detected at 40 and 42 kDa. E, time dependence of the hGH-stimulated association of FAK with CrkII in CHO cells stably transfected with GH receptor cDNA (CHO-GHR 1-638 ). CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and CrkII protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for FAK. The loading control for CrkII is shown in F. The arrowhead marks the position of p125 FAK detected at 125 kDa. The position of the molecular mass standards is indicated on the righthand side. The data presented are representative of at least three separate experiments. pendent association of c-Src with FAK by immunoprecipitation of FAK from cell extracts and subsequent Western blot analysis for c-Src (Fig. 4C). c-Src and FAK were observed to be associated in the unstimulated state (upon longer exposure of the membrane), and hGH stimulation of cells resulted in increased c-Src-FAK association between 2 and 15 min, followed by a decline to 60 min. Similarly we also demonstrated a time-and hGH-dependent association of c-Src with the p130 Cas -CrkII complex by immunoprecipitation of p130 Cas from cell extracts followed by Western blot analysis for c-Src (Fig. 4E). A basal level of association between c-Src and p130 Cas was observed and hGH stimulation of cells resulted in increased c-Src/ p130 Cas association between 2 and 15 min and then followed by a decline to 60 min. Tyrosine phosphorylation of c-Src can either stimulate (Tyr-416) or inhibit (Tyr-527) Src kinase activity (24). We therefore examined Src kinase activity in the cell after hGH stimulation using a specific assay. Cells were therefore cultured to confluence, stimulated with 50 nM hGH, and Src kinase activity measured. hGH stimulation of cells resulted in increased Src kinase activity, maximal at 2 min and followed by a decline to 60 min (Fig. 4G). Thus hGH-stimulated tyrosine phosphorylation of c-Src resulted in an initial increase in Src kinase activity followed by a decline to basal levels. Interestingly though, the hGH-stimulated increase in Src kinase activity was only observed in cells grown to confluence, and no hGH stimulation of Src kinase activity was observed in subconfluent cells (data not shown and Ref. 25).
hGH Stimulates c-Fyn Kinase Activity-c-Fyn has also been reported to associate with FAK and may also participate in the tyrosine phosphorylation of the FAK molecule (26). We demonstrated by Western blot analysis a hGH-and time-dependent increase in the level of c-Fyn protein in phosphotyrosine (PY20) immunoprecipitates from cells treated with hGH (Fig. 1D). c-Fyn protein was identified as a single band of 59 kDa. c-Fyn was observed to be tyrosine-phosphorylated in serum-deprived cells. hGH-stimulated tyrosine phosphorylation of c-Fyn was first observed 2 min after stimulation, and the maximal hGHstimulated tyrosine phosphorylation of c-Fyn occurred between 5 and 15 min after stimulation, with the relative level of c-Fyn tyrosine phosphorylation decreasing at 60 min. We also performed the reciprocal immunoprecipitation and blotting and observed the consistent presence of a major time-and hGH-dependent tyrosine-phosphorylated band of 59 kDa, which comigrated with c-Fyn protein (Fig. 5A). Similarly, increased hGH-dependent tyrosine phosphorylation of c-Fyn was observed first at 2 min, persisted to 30 min, and then declined at 60 min. Immunoprecipitation of CrkII from cell extracts treated with hGH and Western blot analysis for c-Fyn revealed a hGH-dependent association between c-Fyn and the p130 Cas -CrkII complex (Fig. 5C). Some basal level of association between c-Fyn and CrkII was observed (upon longer exposure of the membrane), and hGH stimulation of cells resulted in increased c-Fyn-CrkII association between 2 and 5 min and then followed by a decline to 60 min. We also examined the Fyn kinase activity in the cell after hGH stimulation using a specific assay. hGH stimulation of cells resulted in increased Fyn kinase activity, maximal at 2 min and followed by a decline to ysis for c-Src. The loading control for p130 Cas is shown in F. The arrowhead marks the position of c-Src detected at 60 kDa. G, time dependence of the hGH-stimulated increase in c-Src activity in CHO cells stably transfected with GH receptor cDNA (CHO-GHR 1-638 ). CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and c-Src protein immunoprecipitated. Immunoprecipitated proteins were subjected to in vitro kinase assay as described under "Experimental Procedures." The data are presented as the mean Ϯ S.E. The data presented are representative of at least three separate experiments. CHO-GHR 1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and p130 Cas protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot anal-60 min (Fig. 5E). Interestingly though, like we observed for the hGH-dependent increase in Src kinase activity, the hGH-stimulated increase in Fyn kinase activity was also only observed in cells grown to confluence, and no hGH stimulation of c-Fyn kinase activity was observed in subconfluent cells (data not shown).
Paxillin, Tensin, IRS-1, and PI 3-Kinase Are Present in the GH-stimulated p130 Cas -CrkII Complex-We have reported previously that hGH stimulates the tyrosine phosphorylation and association of tensin with FAK (14). We therefore proceeded to determine whether tensin was present in the CrkII-p130 Cas complex formed by cellular stimulation with hGH. Immunoprecipitation of CrkII from cell extracts and subsequent Western blot analysis for tensin revealed a time-and hGH-dependent association of tensin with the CrkII-p130 Cas complex (Fig. 6A). Tensin was minimally associated with the CrkII-p130 Cas complex in the unstimulated state, and maximal hGH-dependent association of tensin with the CrkII-p130 Cas complex was observed at 5-30 min followed by a decline at 60 min. We have also reported previously that hGH stimulates the tyrosine phosphorylation of paxillin constitutively associated with FAK (14). Since FAK is a component of the CrkII-p130 Cas complex, we therefore proceeded to determine whether paxillin was present in the CrkII-p130 Cas complex formed by cellular stimulation with hGH. CrkII immunoprecipitates subjected to Western blot analysis for paxillin revealed a time-and hGH- dependent association of paxillin with the CrkII-p130 Cas complex. Paxillin was minimally associated with the CrkII-p130 Cas complex in the unstimulated state (visible upon longer exposure of the membrane) and maximal hGH-dependent association of paxillin with the CrkII-p130 Cas complex was observed at 5-30 min (Fig. 6C) followed by a decline at 60 min. CrkII has also been reported to associate with other molecules, including IRS-1 (23), and IRS-1 has been reported previously to be phosphorylated in response to cellular stimulation with GH (27)(28)(29). We therefore examined the association state between IRS-1 and CrkII (Fig. 6E) and demonstrated a time-and hGH-dependent association of IRS-1 with CrkII. IRS-1 was not associated with CrkII in the unstimulated state (or minimal association upon longer exposure of the membrane) and maximal hGH-dependent association of IRS-1 with CrkII was observed at 15 min followed by a decline at 30 and 60 min (Fig. 6F). The p85 regulatory subunit of PI 3-kinase has been reported to associate directly with many of the components of the hGHstimulated CrkII-p130 Cas complex described herein. These include FAK (30), tensin (20), c-Cbl (31) (see below), and IRS-1 (19). We thus reasoned that p85 should be contained in the CrkII-p130 Cas complex formed by cellular stimulation with hGH. We observed a time-and hGH-dependent association of p85 with the CrkII-p130 Cas complex (Fig. 6G). Thus p85 was minimally associated with the CrkII-p130 Cas complex in the unstimulated state, and maximal hGH-dependent association of p85 with the CrkII-p130 Cas complex was observed at 2-5 min followed by a decline to 60 min.
c-Cbl Is Tyrosine-phosphorylated in Response to hGH and Complexes with p130 Cas -CrkII-c-Cbl has been reported to associate with some of the above mentioned proteins (e.g. c-Src, c-Fyn) (32,33), and we have observed the presence of a hGHdependent tyrosine-phosphorylated protein of 120 kDa associated with CrkII. Stimulation of CHO-GHR 1-638 cells with hGH resulted in a time-dependent increase in the level of c-Cbl protein in the phosphotyrosine (PY20) immunoprecipitates (Fig. 1E). c-Cbl protein was identified as a single band of 120 kDa, and some basal level of c-Cbl tyrosine phosphorylation was observed in serum-deprived cells. The maximal hGH-stimulated tyrosine phosphorylation of c-Cbl was observed between 5 and 15 min after stimulation, with the relative level of c-Cbl tyrosine phosphorylation decreasing at 60 min. We also performed the reciprocal immunoprecipitation for c-Cbl protein and examined the level of tyrosine phosphorylation by Western blot analysis with PY20. We observed a time-and hGH-dependent tyrosine-phosphorylated band of 120 kDa, which co-migrated with c-Cbl protein (Fig. 7A). Similarly, increased hGHdependent tyrosine phosphorylation of p120 was observed first at 2 min, was maximal 5-15 min after stimulation, and then declined at 60 min. We also proceeded to determine the association state between c-Cbl and CrkII by immunoprecipitation of CrkII from extracts of cells treated with hGH and subsequent Western blot analysis for c-Cbl (Fig. 7C). c-Cbl was not associated with CrkII in the unstimulated state (or minimally associated upon longer exposure of membrane) and maximal hGH-dependent association of c-Cbl with CrkII was observed at 2-15 min. c-Cbl has been reported to associate with and be phosphorylated by the c-Src and c-Fyn kinases (32,33). Immunoprecipitation of c-Cbl from cell extracts and Western blot analysis for c-Src revealed a time-and hGH-dependent association of c-Src with c-Cbl (Fig. 7E). Some basal level of c-Src was associated with c-Cbl in the unstimulated state, and maximal hGH-dependent association of c-Src with c-Cbl was observed between 2 and 15 min with a decline at 30 and 60 min. Similarly, upon stripping and reprobing of the same membrane, we observed a time-and hGH-dependent association of c-Fyn with c-Cbl (Fig. 7G). Some basal level of c-Fyn was associated with c-Cbl in the unstimulated state, and maximal hGH-dependent association of c-Fyn with c-Cbl was observed between 2 and 15 min followed by a decline at 30 and 60 min.
Nck Is Tyrosine-phosphorylated in Response to hGH and Complexes with p130 Cas -CrkII-Nck has been reported previously to associate with p130 Cas (20) and mediate the downstream activation of JNK/SAPK (34,35). To determine whether hGH could stimulate the tyrosine phosphorylation of Nck, we examined the level of Nck protein in phosphotyrosine immunoprecipitates from hGH-treated cells (Fig. 1F). Nck protein was identified as a single band of 47 kDa. Some basal level of Nck tyrosine phosphorylation was observed in serum-deprived cells. The maximal hGH-stimulated tyrosine phosphorylation of Nck was observed between 2 and 15 min after stimulation, with the relative level of Nck tyrosine phosphorylation decreasing at 60 min. Immunoprecipitation of Nck protein from cell extracts and Western blot analysis with PY20 revealed the consistent presence of a major time-and hGH-dependent tyrosine-phosphorylated band of 47 kDa, which co-migrated with Nck protein (Fig.  8A). Similarly, increased hGH-dependent tyrosine phosphorylation of p47 was observed first at 2 min, was maximal 5-15 after stimulation, and then declined at 60 min. We next proceeded to determine the association state between Nck and p130 Cas -CrkII complex. Immunoprecipitation of CrkII protein from cell extracts treated with hGH and Western blotting analysis for Nck revealed a time-and hGH-dependent association of Nck with CrkII (Fig. 8C). Some basal level of Nck was associated with CrkII in the unstimulated state, and maximal hGHdependent association of Nck with CrkII was observed between 2 and 30 min (Fig. 5C).
C3G, SHC, Grb-2, and Sos-1 Are Components of the p130 Cas -CrkII Complex-C3G has also been reported to associate with CrkII (36) and mediate the downstream activation of JNK/ SAPK (15). SDS-PAGE and Western blotting analysis for C3G after immunoprecipitation of CrkII from cell extracts revealed a constitutive association between C3G and CrkII not dependent on hGH stimulation (Fig. 9C). We also examined the effect of hGH on the association between SHC protein and p130 Cas - CrkII complex. SHC proteins of molecular masses 52 and 66 kDa were associated in a hGH-dependent manner with the p130 Cas -CrkII complex (Fig. 9E). SHC proteins were associated with CrkII in the unstimulated state, and maximal hGH-dependent association of SHC proteins with CrkII was observed at 2-15 min followed by a decline to 60 min. The formation of a FAK-c-Src complex permits c-Src to phosphorylate FAK at tyrosine 925, thereby promoting Grb-2 binding (37). Grb-2 has also been demonstrated to be utilized by GH to mediate downstream signaling to MAP kinase via its association with SHC (38,39). We therefore examined the effect of hGH on the association between Grb-2 and the p130 Cas -CrkII complex. Immunoprecipitation of CrkII from the cell extracts and Western blot analysis for Grb-2 revealed a time-and hGH-dependent association of Grb-2 with CrkII (Fig. 9G). Grb-2 was associated with CrkII in the unstimulated state, and maximal hGH-dependent association of Grb-2 with CrkII was observed at 5-15 min followed by a decline to 60 min. We also examined the effect of hGH on the association between Sos-1 and the p130 Cas -CrkII complex. CrkII was immunoprecipitated from cell extracts, and Western blot analysis of these precipitates for Sos-1 revealed a time-and hGH-dependent association of Sos-1 with CrkII (Fig. 9I). Thus, Sos-1 was associated with CrkII in the unstimulated state, and maximal hGH-dependent association of Sos-1 with CrkII was observed at 5 min followed by disassociation at 15-60 min to below the unstimulated state.
hGH Stimulates JNK/SAPK Activity-Since the hGH-induced CrkII-p130 Cas complex contained two proteins (Nck and C3G) upstream of JNK/SAPK (15,35), we reasoned that hGH should also be able to enhance JNK/SAPK activity. We therefore examined the JNK/SAPK activity in the cell after hGH stimulation using a specific assay. Cells were therefore cultured to confluence, stimulated with hGH, and JNK/SAPK kinase activity measured. hGH stimulation of CHO-GHR 1-638 cells resulted in increased JNK/SAPK kinase activity, maximal at 15 min and followed by a decline to 60 min (Fig. 10). Therefore one possible downstream target of the CrkII-p130 Cas complex formed by cellular stimulation with hGH is JNK/SAPK.
hGH Stimulation of JNK/SAPK Activity Is CrkII-dependent-To determine whether CrkII actually participated in the hGH activation of JNK/SAPK, we utilized NIH3T3 cells stably transfected with either the vector or CrkII cDNA (40). The NIH3T3 cells stably transfected with CrkII cDNA overexpressed CrkII compared with the vector-transfected control cells (40). Both cell lines possessed similar levels of 125 I-hGH binding at the cell surface (data not shown) and were therefore suitable to study the role of CrkII in the hGH stimulation of JNK/SAPK activity. The vector-transfected NIH3T3 cells displayed minimal activation of JNK/SAPK activity in response to 50 nM hGH. In contrast, the NIH3T3 cells overexpressing CrkII exhibited marked increases in JNK/SAPK activity in response to 50 nM hGH (Fig. 11). Thus CrkII is a positive intermediary in the GH signal transduction pathway leading to JNK/SAPK activation.

DISCUSSION
In the present study we have demonstrated that hGH induces the formation of a large multiprotein signaling complex involving proteins that have been previously identified to be activated by GH (FAK, paxillin, tensin (14), IRS-1 (27)(28)(29), SHC (38), Grb-2, Sos-1 (39), and p85 (41)) as well as the proteins newly identified to be involved in GH signal transduction (p130 Cas , Crk-II, c-Src, c-Fyn, c-Cbl, Nck, C3G. Such a large multiprotein signaling complex may also be utilized by other members of the cytokine receptor superfamily to mediate their pleiotropic effects within the cell. Other members of the cytokine receptor superfamily include the receptors for prolactin (PRL), erythropoietin, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, ciliary neurotrophic factor, thrombopoietin, leptin, interleukins (IL) 2-7, IL-9, IL-11, IL-12 (3). The related hormone, PRL, for example, has been reported previously to activate FAK (42), c-Src (43), c-Fyn (44), c-Cbl (45), and p85 (46), and it is likely that the PRL activation of these molecules also includes the formation of the large multiprotein signaling complex centered around p130 Cas -Crk-II. The utilization of similar signaling molecules by members of the cytokine receptor superfamily would explain the enormous redundancy in biological function observed between these molecules (47). Interestingly, though, the hGH-dependent tyrosine phosphorylation of Crk-II and its association with IRS-1 identifies a potential divergence point for signal transduction by hGH and its somatic mediator, IGF-1. According to the somatomedin hypothesis, GH mediates many of its somatic effects by the production of circulating IGF-1, which acts locally in target tissues (3). IGF-1 has been reported to stimulate the tyrosine phosphorylation of Crk-II and subsequent disassociation from IRS-1 (40). We demonstrate here that hGH stimulates the tyrosine phosphorylation and association of Crk-II with IRS-1. How this difference in signal transduction by GH and IGF-1 affects the final response of the cell to stimulation is under investigation. 2 We have described here that both c-Src and c-Fyn kinases are activated in response to hGH. Several of the proteins that we have described in the GH-stimulated signaling complex have been reported to be subtrates for c-Src. These include FAK, p130 Cas (13), and c-Cbl (32). The formation of a FAK-c-Src complex permits c-Src to phosphorylate FAK within the catalytic domain (47) and at tyrosine 925 (48) thus enhancing FAK kinase activity and promoting Grb-2 binding, respectively. The binding of Src family protein tyrosine kinases to the motif surrounding the FAK autophosphorylation site (tyrosine 397) is required for Src-mediated phosphorylation of FAK tyrosine 925 in vivo (48). Overexpression of the c-Src binding site mutant of FAK (Phe-397) inhibits fibronectin-stimulated signaling to MAPK, implying that Src family protein tyrosine kinase binding may be essential for FAK-mediated signaling events (20). Thus one potential pathway for the activation of MAPK by GH is through a FAK-c-Src-Grb-2 complex. FAK can also bind and directly phosphorylate SHC at tyrosine 317 to promote Grb-2 binding and activation of MAPK (49). It has also been demonstrated previously that in addition to the classical MAPK pathway (39), GH utilizes the Grb-2 binding site on the epidermal growth factor receptor in a JAK2 (but not Src)-dependent manner to mediate the downstream activation of MAPK (25). Thus multiple pathways may be utilized by GH to mediate MAPK activation in a manner analogous to integrin signal transduction (49).
In FAK-deficient fibroblasts, p130 Cas is tyrosine-phosphorylated (50) and localized to cell substratum contact sites (51). In Src-deficient fibroblasts p130 Cas is neither tyrosine-phosphorylated (20, 50) nor localized to focal adhesions (51) after integrin stimulation. Thus it appears that c-Src tyrosine-phosphory-lates p130 Cas and localizes p130 Cas to focal adhesions after integrin stimulation. Also it has been demonstrated that both the SH2 and SH3 domains of c-Src bind to specific motifs in p130 Cas (52). Furthermore, it has been postulated that c-Src bound through its SH2 domain to phosphorylated FAK facilitates c-Src SH3 domain interactions with p130 Cas thereby promoting the formation of a ternary complex of FAK, c-Src, and p130 Cas (13). It remains to be determined which kinase (FAK, c-Src, JAK2, or other), or which combination of kinases, are utilized by GH to mediate the tyrosine phosphorylation of p130 Cas .
The molecules involved in the p130 Cas -Crk-II complex are likely to be pivotal for many of the pleiotropic effects of GH on cellular function. We have shown here that hGH can stimulate an increase in JNK/SAPK activity concordant with our demonstration that hGH stimulation of the cell results in the tyrosine phosphorylation of Nck and inclusion of both C3G and Nck in the p130 Cas -CrkII complex. Integrin stimulation has also been been demonstrated previously to promote the binding of Nck to p130 Cas (20). Both C3G and Nck have been reported to be upstream of JNK/SAPK (15,35). A CrkII-C3G complex has been demonstrated to activate JNK/SAPK through a pathway involving the mixed lineage kinase family of proteins (53). Nck connects to the JNK/SAPK pathway by association with SH3 domain-associated protein serine/threonine kinases such as PAK or NIK (34,35). Evidence presented here also indicates that this is the case for hGH, as CrkII overexpression (upstream of C3G (15)) in NIH3T3 cells results in the enhanced hGH-dependent activation of JNK/SAPK activity. The activa- FIG. 11. Effect of CrkII overexpression on the hGH-stimulated increase in JNK/SAPK activity in NIH3T3 cells. NIH3T3 cells stably transfected with CrkII cDNA (NIH3T3-CrkII) and NIH3T3 cells stably transfected with vector (NIH3T3-vector) were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and JNK/SAPK activity determined as described under "Experimental Procedures." The position of the phospho-c-Jun fusion protein is indicated (A, B), and the densitometric analysis of the in vitro JNK/SAPK activity is shown in C. The data presented are representative of at least three separate experiments.