Growth Hormone Stimulates the Tyrosine Phosphorylation and Association of p125 Focal Adhesion Kinase (FAK) with JAK2
FAK IS NOT REQUIRED FOR STAT-MEDIATED TRANSCRIPTION*

Tao Zhu, Eyleen L. K. Goh, and Peter E. LobieDagger

From the Institute of Molecular and Cell Biology and Defense Medical Research Institute, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We have demonstrated that growth hormone (GH) activates focal adhesion kinase (FAK), and this activation results in the tyrosine phosphorylation of two FAK substrates, paxillin and tensin. The activation of FAK is time-dependent (maximal activation at 5-15 min) and dose-dependent (maximal activation at 0.05 nM). FAK and paxillin are constitutively associated in the unstimulated state, remain associated during the stimulation phase, and recruit tyrosine-phosphorylated tensin to the complex after GH stimulation. Half of the carboxyl-terminal region of the GH receptor is dispensable for FAK activation, but FAK activation does require the proline-rich box 1 region of the GH receptor, indicative that FAK is downstream of JAK2. FAK associates with JAK2 but not JAK1 after GH stimulation of cells. Using FAK-replete and FAK-deficient cells, we also show that FAK is not required for STAT-mediated transcriptional activation by GH. The use of FAK in the signal transduction pathway utilized by GH may be central to many of the pleiotropic effects of GH, including cytoskeletal reorganization, cell migration, chemotaxis, mitogenesis, and/or prevention of apoptosis and gene transcription.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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). It has been demonstrated previously that GH utilizes STAT1 and STAT3 (5-7) and STAT5 (8-10). Interestingly, STAT5 is also activated by cell adhesion to the extracellular matrix (ECM), and hormonal activation of STAT5 in mammary epithelial cells is matrix-dependent (11).

Other diverse actions of GH include the stimulation of chemotaxis and migration of monocytic cells (12). Concordantly, we have demonstrated that GH stimulates the reorganization of the actin cytoskeleton in cells with fibroblastic morphology (13). Focal adhesion kinase (p125 FAK) has been postulated to play a central role in the response of the cell to the ECM (for review, see Ref. 14) and in cell morphology and motility (for review, see Ref. 15). For example, overexpression of FAK stimulates cell migration (16), and both FAK-deficient endodermal and mesodermal cells migrate slower than their FAK-replete counterparts (17, 18). To determine the mechanism by which GH exerts its effects on the cytoskeleton and cell motility and to determine the point of interaction of the ECM and cytokine receptor pathways, we have examined the response of FAK and its substrates to cellular stimulation with GH. We show 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. Interestingly, FAK is not required for GH stimulation of STAT5-mediated transcription, indicative that FAK is not the point of convergence of the cytokine receptor and ECM STAT-mediated transcription.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Recombinant human growth hormone (hGH) was a generous gift of Novo-Nordisk (Singapore). Anti-phosphotyrosine mAb (PY20), anti-FAK mAb, anti-phosphotyrosine polyclonal antisera, and anti-paxillin mAb were purchased from Transduction Laboratories (Lexington, KY). Anti-tensin antibodies were obtained both from Transduction Laboratories and Chemicon (Temecula, CA). JAK2 antiserum was from Upstate Biotechnology (Lake Placid, NY). JAK1 antiserum was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-mouse IgG and anti-rabbit IgG were obtained from DAKO (Århus, Denmark). The enhanced chemiluminescence (ECL) kit was purchased from Amersham (Buckinghamshire, U. K.). Protein G, protein A-agarose, and all other reagents were purchased from Sigma. FAK-replete and FAK-deficient mouse embryo fibroblasts (MEF) were a generous gift of Dr. Dusko Ilic (18).

Chinese Hamster Ovary (CHO) Cell Lines Stably Transfected with GH Receptor cDNAs-- Rat GH receptor cDNAs were cloned into an expression plasmid containing an SV40 enhancer and a human metallothionein IIa promoter. The cDNAs were transfected into CHO-K1 cells using Lipofectin together with the pIPB-1 plasmid, which contains a neomycin resistance gene fused to the thymidine kinase promoter (19). Stable integrants were selected using 1,000 µg/ml G418. The complete rat GH receptor cDNA (20) coding for amino acids 1-638 was expressed in CHO4-638 or CHOA-638 cells (19) and will be referred to as CHO-GHR1-638. Both cell clones transfected with the full-length receptor cDNA behaved identically for the experiments described here, although by affinity cross-linking the receptor in CHO4-638 cells has an Mr of 84,000 compared with the expected Mr of 120,000 as observed in CHOA-638 cells (21). The construction of GH receptor cDNA expression plasmids containing a deletion of box 1 (Delta 297-311) and the individual substitution of proline residues 300, 301, 303, and 305 in box 1 for alanine has been described previously (22). These cDNAs were stably transfected into CHO-K1 cells; the Delta 297-311 mutation was expressed in CHO-GHR1-638Delta 297-311 cells, and P300A,P301A, P303A,P305A was expressed in CHO-GHR1-638P300,301,303,305A cells (22). The construction of GH receptor cDNA expression plasmids with stop codons inserted at position 295 (expressed in CHO-GHR1-294 cells) and position 455 (expressed in CHO-GHR1-454 cells) has also been described previously (19). The level of receptor expression for the individual cell clones is comparable between clones and has been described previously (19, 22).

Cell Culture-- CHO cells stably transfected with rat GH receptor cDNA were maintained in Ham's F-12 medium plus 10% v/v fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C as described previously (19). MEF were grown in Dulbecco's modified Eagle's medium and supplemented as above.

Cell Stimulation and Immunoprecipitation-- CHO cells were grown to confluence, incubated for 16 h in serum-free medium, washed twice in serum-free medium, and stimulated with the indicated dose of hGH for the indicated time periods. Cells were lysed at 4 °C in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM sodium orthovanadate, 0.5% Nonidet P-40, 0.1% phenylmethylsulfonyl fluoride) for 30 min with regular vortices. Cell lysates were centrifuged at 14,000 × g for 15 min, and the resulting supernatants were collected and protein concentration determined. 800 µg of protein was used for each immunoprecipitation. Immunoprecipitations were performed routinely by incubating lysates with 2-4 µg/ml of the respective antibody for 2 h at 4 °C. Immunocomplexes were collected either by incubating with 50 µl of protein G-agarose for 1 h (polyclonal antiserum) or by incubating with 5 µg of anti-mouse IgG for 1 h followed by 1-h incubation with protein G-agarose (monoclonal antibodies). Immunoprecipitations were washed three times with ice-cold lysis buffer. The pellet was resuspended in 2 × SDS-sample buffer containing 50 mM Tris, pH 6.8, 2% SDS, 2% beta -mercaptoethanol, and bromphenol blue, boiled for 10 min, and centrifuged at 14,000 × g for 5 min. The supernatant was collected and subjected to 8% SDS-polycrylamide gel electrophoresis (PAGE). Proteins were transferred to nitrocellulose membranes using standard electroblotting procedures.

Western Blot Analysis-- Nitrocellulose membranes were blocked with either 3% insulin-free bovine serum albumin (for phosphotyrosine immunoblotting) or 5% nonfat dry milk (for other immunoblotting) in phosphate-buffered saline with 0.1% Tween 20 for 2 h at 22 °C. Blots were then immunolabeled overnight at 4 °C with PY20 (1:1,000), anti-FAK mAb (1:1,000), anti-paxillin mAb (1:1,000), anti-tensin mAb (1:1,000), JAK2 antiserum (1:500), or JAK1 antiserum (1:500). Immunolabeling was detected by ECL according to the manufacturer's instructions. The blots were stripped and reprobed with the same antibodies used for their immunoprecipitation to ensure equal loading of the immunoprecipitated protein. Blots were stripped by incubation for 30 min at 50 °C in a solution containing 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 0.7% beta -mercaptoethanol. Blots were then washed for 30 min with several changes of PBS and 0.1% Tween 20 at room temperature. Efficacy of stripping was determined by reexposure of the membranes to ECL. Thereafter, blots were reblocked and immunolabeled as described above.

Transient Transfection and Reporter Assay-- Wild type MEF and MEF deficient in FAK (17) were cultured to confluence in six-well plates. Transient transfection was performed in serum-free Dulbecco's modified Eagle's medium with DOTAP according to the manufacturer's instructions. 1 µg of reporter plasmid (SPI-GLE1 or c-fos-SIE-CAT) and 1 µg of pSV2-LUC ± 1 µg of GH receptor expression plasmid were transfected per well. Cells were incubated with DOTAP/DNA for 12 h before the medium was changed to serum-free Dulbecco's modified Eagle's medium or serum-free Dulbecco's modified Eagle's medium containing 50 nM hGH. After an additional 24 h, cells were washed in phosphate-buffered saline and scraped into lysis buffer. The protein contents of the samples were normalized, and chloramphenicol acetyltransferase (CAT) and luciferase (LUC) assays were performed as described previously (8). Results were normalized to the level of luciferase to control for transfection efficiency and calculated as the ratio of stimulated to unstimulated (non-hormone-treated) cells.

Statistics and Presentation of Data-- All experiments were repeated at least three times, usually five to seven times. The figures presented for Western blot analyses are representative of multiple experiments. The text under "Results" summarizes the outcome from multiple Western blot analyses; consequently, the text (e.g. description of the time course of phosphorylation and dephosphorylation) may not correspond exactly to the actual figure presented. All numerical data are expressed as mean ± S.D. Data were analyzed using the two-tailed t test or analysis of variance.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

To determine if GH could stimulate the tyrosine phosphorylation of FAK we first treated CHO-GHR1-638 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 FAK revealed a GH- and time-dependent increase in the level of FAK protein in the phosphotyrosine immunoprecipitates (Fig. 1A). FAK protein was identified as a single band of 125 kDa. Some basal level of FAK tyrosine phosphorylation was observed in serum-deprived cells. The maximal GH-stimulated tyrosine phosphorylation of FAK was observed between 10 and 15 min after stimulation, with the relative level of FAK tyrosine phosphorylation decreasing at 60 min. We next immunoprecipitated FAK from cell extracts of CHO-GHR1-638 cells stimulated for 0-60 min with 50 nM hGH and examined the level of tyrosine phosphorylation by Western blot analysis with PY20. We observed the consistent presence of two major time- and hGH-dependent tyrosine-phosphorylated bands of 125 and 215 kDa, respectively (Fig. 2A). The 125-kDa band co-migrated with FAK protein, and the 215-kDa protein co-migrated with tensin protein (see below). Increased hGH-dependent tyrosine phosphorylation of FAK was apparent at 5 min and was maximal from 10 to 15 min followed by a decline at 60 min. Some basal level of FAK tyrosine phosphorylation was also evident upon longer exposure of the membrane. Equal amounts of FAK protein were immunoprecipitated at all time points as indicated by the loading control (Fig. 2B). Similarly, increased hGH-dependent tyrosine phosphorylation of p215 was observed first at 5 min, was maximal 10-15 after stimulation, and then declined at 60 min. Some other tyrosine-phosphorylated proteins of different molecular mass were also present (upon longer exposure of the membranes; data not shown) in the FAK immunoprecipitates, albeit at lower levels than p125 and p215. Such proteins possessed molecular masses of 85 kDa (and may represent the p85 subunit of PI-3 kinase (23) and 128 kDa (see below; identified as JAK2). We also examined the dose dependence of the hGH stimulation of FAK tyrosine phosphorylation. We therefore stimulated CHO-GHR1-638 cells with 0, 0.005, 0.05, 0.5, 5, and 50 nM hGH, prepared cell extracts, and immunoprecipitated FAK from the cell extracts. SDS-PAGE and Western blot analysis (PY20) of the FAK immunoprecipitates again revealed GH-stimulated tyrosine phosphorylation of p125 and p215. Human GH-stimulated tyrosine phosphorylation was first observed at 0.005 nM hGH, and maximal tyrosine phosphorylation was observed at 0.05-0.5 nM hGH (Fig. 2C). Some diminution of the hGH response was observed at higher doses presumably concordant with the receptor dimerization theory of GH signal transduction (24). We continued to use 50 nM hGH for consistency of cellular responses.


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Fig. 1.   Human GH stimulation of tyrosine phosphorylation of FAK, paxillin, and tensin in CHO cells stably transfected with GH receptor cDNA (CHO-GHR1-638). CHO-GHR1-638 cells were stimulated with 50 nM hGH for the indicated time periods. Cell extracts were prepared and tyrosine phosphorylated proteins precipitated with PY20 as described under "Experimental Procedures." Immunoprecipitates were subjected to SDS-PAGE and Western blot analysis for FAK (A), paxillin (B), and tensin (C). The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments.


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Fig. 2.   Time and dose dependence of the hGH-stimulated tyrosine phosphorylation of FAK in CHO cells stably transfected with GH receptor cDNA (CHO-GHR1-638). A, CHO-GHR1-638 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 tyrosine phosphorylation with the use of PY20. The loading control for FAK is shown in B. Arrowheads mark the positions of the two major tyrosine-phosphorylated bands detected at 125 and 215 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments. C, CHO-GHR1-638 cells were stimulated with the indicated concentrations of hGH for 10 min, cell extracts prepared, and FAK 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 FAK is shown in D. Arrowheads mark the positions of the two major tyrosine-phosphorylated bands detected at 125 and 215 kDa. The positions of the molecular weight standards are indicated on the right. The data are representative of at least three separate experiments.

FAK has been reported to be constitutively associated with paxillin, and paxillin is a major substrate for FAK both in vitro and in vivo (25, 26). To first determine if GH could stimulate the tyrosine phosphorylation of paxillin we treated CHO-GHR1-638 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 paxillin revealed a time- and hGH-dependent increase in the level of paxillin protein in the phosphotyrosine immunoprecipitates (Fig. 1B). Paxillin protein was identified as a band of between 66 and 68 kDa. Some basal level of paxillin tyrosine phosphorylation was observed in serum-deprived cells. The maximal hGH-stimulated tyrosine phosphorylation of paxillin was observed between 10 and 30 min after stimulation with the relative level of paxillin tyrosine phosphorylation decreasing at 60 min. We next immunoprecipitated paxillin from extracts of CHO-GHR1-638 cells stimulated for 0-60 min with 50 nM hGH and examined the level of tyrosine phosphorylation by Western blot analysis with PY20. Because paxillin runs close to the heavy chain of the IgG molecule on SDS-PAGE, we first verified the identity of the paxillin band (Fig. 3A). Upon GH stimulation of CHO-GHR1-638 cells we observed the appearance of a heavily tyrosine-phosphorylated band at 66-68 kDa in paxillin immunoprecipitates, although basal tyrosine phosphorylation of the 66-68-kDa protein was also evident. No such tyrosine-phosphorylated protein band was evident when a control monoclonal antibody (c-Jun) was used, and use of this control monoclonal antibody allowed identification of the IgG heavy chain as a separate protein from the tyrosine-phosphorylated 66-68-kDa band (Fig. 3A). Further, the tyrosine-phosphorylated 66-68-kDa protein co-migrated with paxillin, when paxillin immunoprecipitates were electrophoresed on the same polyacrylamide gel and immunoblotted for paxillin. Also, co-migration of the tyrosine-phosphorylated band at 66-68 kDa with paxillin protein was observed upon stripping of the membrane and subsequent reblotting of the membrane for paxillin loading control (for example, see Fig. 3B). Increased hGH-dependent tyrosine phosphorylation of paxillin was apparent at 5 min and was maximal from 10 to 30 min followed by a decline at 60 min (Fig. 3B). Some basal tyrosine phosphorylation of paxillin was observed in cells in the unstimulated state. When SDS-PAGE was allowed to proceed for a longer time such that the large molecular mass proteins could enter the running gel, the GH- and time-dependent tyrosine phosphorylation of proteins of 125 and 215 kDa was observed (Fig. 3D). The 125-kDa tyrosine-phosphorylated protein co-migrated with FAK protein. Increased hGH-dependent tyrosine phosphorylation of FAK was observed first at 5 min, was maximal 10-30 after stimulation, and then declined at 60 min. The 215-kDa protein was identified as tensin (see below). We also examined the dose dependence of the hGH stimulation of tyrosine phosphorylation of the paxillin-associated 125-kDa (FAK) and the 215-kDa (tensin) proteins. We therefore stimulated CHO-GHR1-638 cells with 0, 0.005, 0.05, 0.5, 5, and 50 nM hGH, prepared cell extracts, and immunoprecipitated paxillin from the cell extracts. SDS-PAGE and Western blot analysis (PY20) of the paxillin immunoprecipitates again revealed GH-stimulated tyrosine phosphorylation of p125 and p215 (Fig. 3F). Maximal hGH-stimulated tyrosine phosphorylation was observed at 0.05-0.5 nM hGH. Again, some diminution of the hGH response was observed at higher doses of hGH. We next proceeded to determine the association state between FAK and paxillin. We therefore treated CHO-GHR1-638 cells for 0-60 min with 50 nM hGH, prepared cell extracts, and immunoprecipitated FAK from the cell extracts. Subsequent SDS-PAGE and Western blotting analysis for paxillin revealed a constitutive association between FAK and paxillin (66-68 kDa) which was not dependent upon hormonal stimulation (Fig. 4).


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Fig. 3.   Time and dose dependence of the hGH-stimulated tyrosine phosphorylation of paxillin and paxillin-associated proteins in CHO cells stably transfected with GH receptor cDNA (CHO-GHR1-638). A, CHO-GHR1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and paxillin protein immunoprecipitated (lanes 1, 2, 5, and 6) or a control antibody (c-jun) used (lanes 3 and 4) as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for tyrosine phosphorylation with the use of PY20 (lanes 1-4) or paxillin protein (lanes 5 and 6). The arrowheads mark the positions of the tyrosine-phosphorylated band detected at 66-68 kDa and the IgG heavy chain. The position of the molecular mass standard is indicated on the right. The data are representative of at least three separate experiments. B, CHO-GHR1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and paxillin 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 paxillin is shown in C. The arrowhead marks the position of the tyrosine-phosphorylated band detected at 66-68 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments. D, Western blot analysis performed similarly to B but run such that large molecular mass proteins can enter the SDS-PAGE running gel easily. Arrowheads mark the positions of the two major tyrosine-phosphorylated bands detected at 125 and 215 kDa. The loading control for paxillin is shown in E. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments. F, CHO-GHR1-638 cells were stimulated with the indicated concentrations of hGH for 10 min, cell extracts prepared, and paxillin-associated proteins 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 paxillin is shown in G. Arrowheads mark the positions of the two major tyrosine-phosphorylated bands detected at 125 and 215 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments.


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Fig. 4.   FAK and paxillin are constitutively associated in CHO-GHR1-638 cells. CHO-GHR1-638 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 paxillin protein. The loading control for FAK is shown in B. Paxillin is indicated as a broad protein band of 66-68 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments.

In the above experiments hGH stimulated the tyrosine phosphorylation of a protein band of 215 kDa, observed in both FAK and paxillin immunoprecipitates. This protein is likely to be tensin because tensin is a 215-kDa protein substrate of FAK (27). To determine first if hGH could stimulate the tyrosine phosphorylation of tensin we treated CHO-GHR1-638 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 tensin revealed a GH- and time-dependent increase in the level of tensin protein in the phosphotyrosine immunoprecipitates (Fig. 1C). Tensin protein was identified as a band of 215 kDa. Some basal level of tensin tyrosine phosphorylation was observed in serum-deprived cells. The maximal GH-stimulated tyrosine phosphorylation of tensin was observed between 10 and 30 min after stimulation with the relative level of tensin tyrosine phosphorylation decreasing at 60 min. We next immunoprecipitated tensin from cell extracts of CHO-GHR1-638 cells stimulated for 0-60 min with 50 nM hGH and examined the level of tyrosine phosphorylation by Western blot analysis with PY20. We observed the presence of two time- and hGH-dependent tyrosine-phosphorylated bands of 125 and 215 kDa, respectively (Fig. 5A). The 125-kDa band co-migrated with FAK protein as expected, and the 215-kDa protein co-migrated with tensin protein. Paxillin could not be identified definitively because of its proximity to the IgG heavy chain. Increased hGH-dependent tyrosine phosphorylation of tensin was apparent at 5 min and was maximal from 10 to 30 min followed by a decline at 60 min. Similarly, increased hGH-dependent tyrosine phosphorylation of FAK was observed first at 5 min, was maximal 10 min after stimulation, and then declined to 60 min. We next proceeded to determine the association state between FAK and tensin. We therefore treated CHO-GHR1-638 cells for 0-60 min with 50 nM hGH, prepared cell extracts, and immunoprecipitated FAK from the cell extracts. Subsequent SDS-PAGE and Western blotting analysis for tensin revealed a time- and hGH-dependent association of tensin with FAK. Tensin was minimally associated with FAK in the unstimulated state, and maximal hGH-dependent association of tensin with FAK was observed between 10 and 30 min (Fig. 5C).


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Fig. 5.   Time dependence of the hGH-stimulated tyrosine phosphorylation of tensin and association of tensin with FAK in CHO cells stably transfected with GH receptor cDNA (CHO-GHR1-638). A, CHO-GHR1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and tensin 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 tensin is shown in B. Arrowheads mark the positions of the two major tyrosine-phosphorylated bands detected at 125 and 215 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments. C, CHO-GHR1-638 cells were stimulated for the indicated time periods with 50 nM hGH, cell extracts prepared, and FAK protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for tensin protein. The loading control for FAK is shown in D. Arrowheads mark the positions of tensin at 215 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments.

We proceeded to determine the GH receptor regions required for FAK activation. We therefore utilized well characterized CHO cell clones stably expressing the wild type receptor (CHO-GHR1-638), a receptor truncation containing only 5 of 349 amino acids in the intracellular domain (CHO-GHR1-294), a receptor truncation containing 184 of 349 amino acids in the intracellular domain (CHO-GHR1-454), a receptor mutation in which the proline-rich box 1 region had been deleted (CHO-GHR1-638Delta 297-311), and a receptor mutation in which the individual proline residues of box 1 had been converted to alanine (CHO-GHR1-638P300,301,303,305A) (Fig. 6A). We therefore stimulated each cell line with 50 nM hGH, prepared cell extracts, and examined FAK tyrosine phosphorylation by Western blot analysis. Of these receptor mutations, only CHO-GHR1-638 and CHO-GHR1-454 displayed a hormone-dependent activation of FAK tyrosine phosphorylation (Fig. 6B). Parental untransfected CHO cells also did not display hGH-dependent activation of FAK. The proline-rich box 1 region of the receptor is required for the association and activation of JAK1 and JAK2 by GH (3). This association is direct and is the initial event in GH signal transduction into the cell; no GH-dependent tyrosine phosphorylation in the cell is achieved with receptor mutations lacking box 1 (22 and data not shown). Thus it is apparent that the GH activation of FAK requires the proline-rich box 1 region of the GH receptor.


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Fig. 6.   GH receptor regions required for hGH stimulation of FAK tyrosine phosphorylation in CHO cells stably transfected with wild type GH receptor and mutated GH receptor cDNA. A, schematic diagram of the GH receptor and the various GH receptor mutations/deletions used. The wild type receptor has the extracellular, transmembrane, and intracellular regions indicated. GHR1-294 expresses only 5 of 349 amino acids in the intracellular domain, GHR1-638Delta 297-311 has the proline-rich region (box 1) deleted, GHR1-638P300,301,303,305A has the individual proline residues in box 1 mutated to alanine, and GHR1-454 expresses 184 of 349 amino acids in the intracellular domain. B, untransfected parental CHO cells and CHO cells expressing the various GH receptor mutations or deletions were stimulated with 50 nM hGH for 10 min, cell extracts prepared, and FAK 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. Arrowheads mark the positions of the two major tyrosine-phosphorylated bands detected at 125 and 215 kDa. The loading control for FAK is shown in C. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments.

Because the box 1 region of the receptor is required for GH-dependent JAK association and activation, we examined a possible association between FAK and either JAK1 or JAK2. JAK2 is the major GH-activated member of the JAK family. We therefore treated CHO-GHR1-638 cells for 0-60 min with 50 nM hGH, prepared cell extracts, and immunoprecipitated FAK from the cell extracts. Subsequent SDS-PAGE and Western blotting analysis for JAK2 revealed a time- and hGH-dependent association of FAK with JAK2 (Fig. 7A). Association between FAK and JAK2 was apparent at 5 min, maximal at 10-30 min after hGH stimulation, and diminished 60 min after hGH stimulation. GH has also been demonstrated to activate JAK1, but the activation of JAK1 is minimal compared with the GH-stimulated activation of JAK2 (28, 29). No apparent association between JAK1 and FAK existed because JAK1 could not be detected in FAK immunoprecipitates despite the ability of the JAK1 antibody to recognize JAK1 by Western blot analysis in JAK1 immunoprecipitates of the cell lysates (data not shown). Conversely, we also showed the JAK2-FAK association by immunoprecipitation with JAK2 and immunoblotting with FAK (Fig. 7C).


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Fig. 7.   Time dependence of the hGH-stimulated association between JAK2 and FAK in CHO cells stably transfected with GH receptor cDNA (CHO-GHR1-638). A, CHO-GHR1-638 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 JAK2 protein. The loading control for FAK is shown in B. Arrowhead marks the position of JAK2 protein at 128 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments. C, CHO-GHR1-638 cells were stimulated with 50 nM hGH for the indicated time periods, cell extracts prepared, and JAK2 protein immunoprecipitated as described under "Experimental Procedures." Immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis for FAK protein. The loading control for JAK2 is shown in D. Arrowhead marks the position of FAK protein at 125 kDa. The positions of the molecular mass standards are indicated on the right. The data are representative of at least three separate experiments.

GH utilizes the JAK-STAT pathway for transcriptional regulation. To determine if the GH-dependent tyrosine phosphorylation of FAK is required for GH-dependent STAT-mediated transcription we examined the transcriptional response in FAK-deficient and FAK-replete cells. For this we utilized MEF from normal and FAK knockout mice and measured the transcriptional response mediated by STAT1 and STAT3 (c-fos-SIE-CAT) and STAT5 (SPI-GLE1-CAT) by reporter assay. Pooled MEF did not possess a response to hGH either through c-fos-SIE-CAT or SPI-GLE1-CAT in the absence of transfected rat GH receptor cDNA (Fig. 8A). Concomitant transient transfection of rat GH receptor cDNA enabled a hGH-dependent STAT5-mediated response through SPI-GLE1-CAT to an extent similar to that observed in other cell lines. No significant hGH stimulation of reporter activity from c-fos-SIE-CAT was observed in MEF even upon co-transfection of the GH receptor cDNA. Pooled MEF from FAK-deficient mice transiently transfected with GH receptor cDNA also responded to hGH stimulation with a response through SPI-GLE1-CAT similar to that observed with the wild type MEF. Such results were also observed with individual cell clones from FAK-replete and FAK-deficient mice (Fig. 8B), indicative that FAK is not required for GH-stimulated STAT-mediated transcriptional activation. No significant response of c-fos-SIE-CAT to GH was obtained in MEF derived from FAK-deficient mice.


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Fig. 8.   A, hGH stimulation of STAT-mediated transcription in pooled FAK-deficient and FAK-replete mouse embryo fibroblasts. Pooled MEF were grown to confluence and transiently transfected with reporter plasmids for either STAT1 and STAT3 (c-fos-SIE-CAT) or STAT5 (SPI-GLE1-CAT) and pSV2-LUC ± GH receptor cDNA as described under "Experimental Procedures." Cells were treated with 50 nM hGH and processed for CAT and luciferase activity as described. Vehicle was used as control. Results represent the mean ± S.D. of triplicate estimations. The result are representative of at least three separate experiments. B, hGH stimulation of STAT-mediated transcription in individual clones of FAK-deficient and FAK-replete MEF. MEF were grown to confluence and transiently transfected with the reporter plasmid for STAT5 (SPI-GLE1-CAT) and pSV2-LUC ± GH receptor cDNA as described under "Experimental Procedures." Cells were treated with 50 nM hGH and processed for CAT and luciferase activity as described. Vehicle was used as control. Results represent the mean ± S.D. of triplicate estimations. The results are representative of at least three separate experiments.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We have demonstrated that p125 FAK and at least two of its substrates are tyrosine-phosphorylated in response to cellular stimulation by GH. We have also placed FAK as a component of the JAK pathway and have therefore described a novel mechanism for the cross-talk between members of the cytokine receptor superfamily and signal transduction by the ECM.

We have provided a possible mechanism for the interaction of cytokine signaling pathways and those utilized by the ECM. Exactly how these two pathways interact is an interesting question. For example, expression of beta -casein in mammary epithelial cells is regulated by lactogenic hormones (prolactin, GH) and the ECM (30). In the absence of the ECM, cells are flat and do not produce beta -casein. Use of ECM results in cell rounding and clustering, which are apparently necessary for the cell to respond to prolactin. Both prolactin and GH regulate the expression of beta -casein (31) and beta -lactoglobulin through STAT5 (11), and STAT5 is required for the ECM dependence of beta -lactoglobulin expression (11). In this regard it is interesting that FAK is not required for STAT5 activation as it may have provided the common link for hormone and ECM activation of certain transcriptional events such as those required for milk protein production. Further evidence that FAK or a FAK-related protein is not required for STAT5 activation is the ability of GH to activate STAT5 normally in the presence of cytochalasin B or cytochalasin D (13). Cytochalasins destroy the integrity of the actin cytoskeleton, which is required for FAK activation and function (32, 33).

The use of FAK by GH for signal transduction permits the GH signal to be propagated through multiple alternate transduction pathways. Tyrosine phosphorylation of the p85 subunit of PI-3 kinase is regulated by cell adhesion in vivo, and it can be phosphorylated by FAK in vitro (23) suggestive that PI-3 kinase may be a substrate of FAK in vivo. The association of FAK and PI-3 kinase is direct and dependent on FAK autophosphorylation (23, 34). GH has been demonstrated to promote the association of the p85 subunit of PI-3 kinase with IRS-1 (35, 36), IRS-2 (37), and JAK2 (38) and to increase the PI-3 kinase activity associated with IRS-1 (35). It is therefore possible that GH may utilize the FAK-PI-3 kinase pathway to increase phosphatidylinositol 3,4,5-trisphosphate levels within the cell as well as the IRS-PI-3 kinase pathway. Such potential utilization of two alternate pathways to activate the same kinase may permit the use of PI-3 kinase for distinct cellular purposes. For example, the activation of PI-3 kinase via IRS may be involved in GH stimulation of metabolic events such as lipogenesis (39), whereas activation of PI-3 kinase via FAK may regulate GH-stimulated cytoskeletal reorganization (13). Such alternate use of pathways would allow the cell to respond precisely to hormonal stimuli dependent on cell type and differentiation status. The use of cells specifically deficient in either IRS-1/IRS-2 and FAK will be instrumental in resolving these hypotheses. GH has also been demonstrated to stimulate mitogen-activated protein kinase activity (19, 40, 41) and the association of SHC and Grb2 with JAK2 (42, 43). JAK2 is required for GH stimulation of mitogen-activated protein kinase activity (22, 44) and also for Ras and Raf activation, which mediates GH activation of mitogen-activated protein kinase (44). Tyr925 of FAK is phosphorylated by c-Src and serves as a binding site for the Grb2·Sos complex both in vivo and in vitro (34). It is therefore possible that FAK is an upstream intermediary in the GH stimulation of the mitogen-activated protein kinase pathway. Similarly, platelet-derived growth factor stimulation of FAK has been reported to be PI-3 kinase-dependent (45), and therefore FAK may complex with JAK via Tec and PI-3 kinase (46). Studies using FAK-deficient cells stably transfected with GH receptor cDNA should be useful in delineating the role of FAK in the GH activation of various signal transduction pathways.

The GH-stimulated tyrosine phosphorylation of the actin-binding protein tensin (27) may provide a possible explanation for the observed effects of GH on the actin cytoskeleton (13). Actin filament polymerization involves exchange of subunits of filament ends, which can be controlled by other proteins that bind actin filaments and inhibit subunit addition or loss at the ends (47). Purified tensin cross-links actin filaments and retards actin assembly by barbed end capping (27). Three regions from tensin interact with actin, two of these do not alter the kinetics of actin assembly, and the third retards actin polymerization (27). How the GH tyrosine phosphorylation of tensin alters the polymerization/depolymerization rates of actin requires further investigation of the function of tensin in actin cytoskeletal dynamics. In any case, we have provided a direct link from the GH receptor via JAK, FAK, and tensin to the actin cytoskeleton. It is also interesting that the GH-induced reorganization of the actin cytoskeleton requires PI3-kinase activity (13). Platelet-derived growth factor has recently been described to stimulate the association of PI3-kinase and tensin via the SH2 domain of tensin (48), and such a similar potential association induced by GH may explain the PI3-kinase dependence of the GH-induced cytoskeletal reorganization.

The FAK-paxillin signaling complex also contains other proteins that have not been described here (for review, see Ref. 49). An early step in FAK activation involves targetting of Src family kinases to FAK, and the Src kinases (c-Src and c-Fyn) are responsible for further tyrosine phosphorylation of FAK (50; for review, see Ref. 49). GH has previously been reported to stimulate the tyrosine phosphorylation of c-Fyn (51). FAK also associates with p130 Cas, and Cas and paxillin recruit other SH2-containing signaling proteins such as c-Crk, which couple to guanine nucletide exchangers (Sos and C3G) for Ras or Rap1 (for review, see Ref. 50). Further analysis of the components of the GH-stimulated FAK signaling complex is currently in progress. In any case, FAK is likely to be pivotal in mediating many of the pleiotropic cellular effects attributed to GH.

In conclusion we have demonstrated that GH induces the tyrosine phosphorylation of FAK and its substrates in a JAK-dependent manner. The use of FAK in the signal transduction pathway utilized by GH may be central to many of the pleiotropic effects of GH, including cytoskeletal reorganization, cell migration, chemotaxis, mitogenesis, and/or prevention of apoptosis and gene transcription.

    FOOTNOTES

* This work was supported by an Institute of Molecular and Cell Biology (Singapore) project grant (to P. E. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 65-874-7847; Fax: 65-779-1117; E-mail: mcbpel{at}mcbsgs1.imcb.nus.edu.sg.

1 The abbreviations used are: GH, growth hormone; ECM, extracellular matrix; FAK, focal adhesion kinase; hGH, human growth hormone; mAb, monoclonal antibody; ECL, enhanced chemiluminescence; MEF, mouse embryo fibroblasts; CHO, Chinese hamster ovary; GHR, growth hormone receptor; PAGE, polyacrylamide gel electrophoresis; DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate; CAT, chloramphenicol acetyltransferase; LUC, luciferase; PI-3 kinase, phosphatidylinositol 3-kinase; IRS, insulin receptor substrate.

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Abstract
Introduction
Procedures
Results
Discussion
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