©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth Hormone-promoted Tyrosyl Phosphorylation of SHC Proteins and SHC Association with Grb2 (*)

(Received for publication, October 18, 1994; and in revised form, December 27, 1994)

Joyce VanderKuur (1)(§) Giovanna Allevato (2) Nils Billestrup (2) Gunnar Norstedt (3) Christin Carter-Su (1)(¶)

From the  (1)Department of Physiology, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, the (2)Hagedorn Research Laboratory, Neils Steensenevej 6, DK-2820 Gentofte, Denmark, and the (3)Center for Biotechnology, Karolinska, Novum, 141 57, Huddinge, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Growth hormone (GH) has been shown to stimulate the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal regulated kinases) 1 and 2. One pathway by which ERKs 1 and 2 are activated by tyrosine kinases involves the Src homology (SH)-2 containing proteins SHC and Grb2. To gain insight into pathways coupling GH receptor (GHR) to MAP kinase activation and signaling molecules that might interact with GHR and its associated tyrosine kinase JAK2, we examined whether SHC and Grb2 proteins serve as signaling molecules for GH. Human GH was shown to promote the rapid tyrosyl phosphorylation of 66-, 52-, and 46-kDa SHC proteins in 3T3-F442A fibroblasts. GH also promoted binding of GHR and JAK2 to the SH2 domain of 46/52-kDa SHC protein fused to glutathione S-transferase (GST). Constitutively phosphorylated JAK2, from COS-7 cells transiently transfected with murine JAK2 cDNA, bound to SHC SH2-GST fusion protein, demonstrating that the SHC SH2 domain can bind tyrosyl-phosphorylated JAK2 in the absence of GHR. Regions of GHR required for GH-dependent tyrosyl phosphorylation of SHC were examined using Chinese hamster ovary cells expressing mutated rat GHR. In cells expressing GHR1-638 and GHR1-638(Y333,338F), GH stimulated phosphorylation of all 3 SHC proteins whereas GH stimulated phosphorylation of only the 66- and 52-kDa SHC proteins in cells expressing GHR1-454. GH had no effect on SHC phosphorylation in cells expressing GHR1-294 or GHRDeltaP, the latter lacking amino acids 297-311 containing the proline-rich motif required for JAK2 activation by GH. In contrast to SHC, Grb2 appeared not to interact directly with GHR or JAK2. However, Grb2 was shown to associate rapidly with SHC proteins in a GH-dependent manner. These findings suggest that GH stimulates: 1) the association of SHC proteins with JAK2bulletGHR complexes via the SHC-SH2 domain, 2) tyrosyl phosphorylation of SHC proteins, and 3) subsequent Grb2 association with SHC proteins. These events are likely to be early events in GH activation of MAP kinases and possibly of other responses to GH.


INTRODUCTION

Our laboratory has shown that GH (^1)binds to the GH receptor (GHR) and activates the GHR-associated tyrosine kinase JAK2, whereupon both GHR and JAK2 become tyrosyl phosphorylated(1) . However, the signaling pathways initiated by the activated GHRbulletJAK2 complex that lead to GH-dependent changes in gene expression, metabolism, cellular differentiation, and body growth are poorly understood(2, 3) . One class of signaling molecules known to be activated by GH (4, 5, 6, 7) is the mitogen-activated protein kinases, commonly referred to as MAP kinases or extracellular signal regulated kinases (ERKs). MAP kinases are believed to play a pivotal role in the regulation of cellular growth and differentiation (8) and have been shown to phosphorylate a number of proteins, including other protein kinases, cytoplasmic phospholipase A(2), cytoskeletal proteins, and transcription factors (9, 10, 11, 12, 13) . Therefore, MAP kinases are likely to represent important signaling molecules for GH.

A well studied pathway leading from membrane receptor tyrosine kinases to MAP kinase activation involves SHC and Grb2 proteins(14, 15, 16) . Two SHC proteins, p46 and p52, have been cloned and found to be differential splice products. A third protein (p66) is recognized by SHC antibodies made to a C-terminal peptide of the 46/52-kDa proteins. It is translated from a distinct transcript. SHC proteins contain a src homology (SH)-2 and a collagen-like domain(17) . Like other SH2-containing proteins(18, 19, 20) , SHC proteins have been shown to bind to phosphotyrosine-containing sequences, including sequences in ligand-activated tyrosine kinase receptors. SHC proteins themselves are substrates for tyrosine kinases(21) . Upon tyrosyl phosphorylation, they serve as linker, or adaptor, proteins for other SH2-containing proteins in tyrosine kinase signal transduction pathways.

Grb2 is a 23-25-kDa protein containing an SH2 domain between two SH3 domains(22) . Unlike SHC proteins, Grb2 is not tyrosyl phosphorylated in response to growth factor stimulation. Some tyrosyl-phosphorylated membrane receptor tyrosine kinases have been shown to interact directly with Grb2, while others have been shown to bind and tyrosyl phosphorylate SHC which in turn binds Grb2(15, 23, 24, 25) . Recruitment of Grb2 in response to ligand binding is believed to initiate one pathway that leads to the activation of MAP kinases. In essence, Grb2 has been shown to interact with the mammalian homolog of the Drosophila gene product son of sevenless, a guanine nucleotide exchange factor which activates the small GTP binding protein, RAS. RAS, in turn, activates the serine/threonine kinase Raf. Raf phosphorylates and activates the mixed function serine/threonine/tyrosine kinase MEK which then phosphorylates and activates the MAP kinases designated ERKs 1 and 2(26) . It has recently been suggested that Grb2 may also serve as a linker protein in other signaling pathways(25, 27) .

In this work, we examined whether GH recruits SHC and Grb2. The mechanism of interaction of these proteins with GHR and JAK2 was also assessed. The results of this study provide insight into signaling molecules that interact with GHR and its associated tyrosine kinase JAK2, and signaling pathway(s) leading from GHR/JAK2 to MAP kinase activation. Involvement of JAK2 suggests that other ligands that activate JAK2 are likely to activate MAP kinases by a pathway including SHC and Grb2 proteins.


EXPERIMENTAL PROCEDURES

Materials

Recombinant DNA-derived 22,000 dalton hGH was a gift of Eli Lilly Co. Recombinant protein A-agarose was from Repligen, and Protein assay (BCA) was from Pierce. Triton X-100, aprotinin, and leupeptin were purchased from Boehringer Mannheim. Chicken egg ovalbumin and glutathione-agarose were purchased from Sigma, prestained molecular weight standards were from Life Technologies, Inc., and nitrocellulose membranes were from Schleicher & Schuell. The enhanced chemiluminescence (ECL) detection system, anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase, protein A-conjugated to horseradish peroxidase, and x-ray film were from Amersham. Fusion proteins encoding the C terminus of glutathione S-transferase (GST) fused to either human Grb2 (22) or human SHC SH2 domain (16) were a gift from Drs. A Saltiel and S. Decker (Parke-Davis, Ann Arbor, MI). Expression plasmids containing the full-length cDNA for murine JAK2 cloned into the SmaI site of pRK5 and for murine JAK2(K882E) (in which glutamic acid replaced the lysine at position 882) cloned into the NotI and EcoRV sites of pRK5 were gifts from Drs. B. Witthuhn and J. Ihle (St. Jude Children's Research Hospital, Memphis, TN).

Mutagenesis, Transfection, and Cell Culture

The stock of 3T3-F442A cells were a kind gift of H. Green (Harvard University, Boston, MA) and were cultured as described previously(28) . CHO cells were cotransfected with plasmids pLM108 and pIBP-1(6, 29) . Plasmid pIBP-1 contains a thymidine kinase promoter fused to the bacterial neomycin phosphotransferase gene conferring G418 resistance. Plasmid pLM108 contains the simian virus 40 enhancer and the Zn-inducible human metallothionein IIa promoter driving the expression of the cDNA coding for full-length rat liver GHR, the same cDNA with termination codons replacing the lysine codons 455, 381, 319, and 295, the same cDNA with phenylalanines replacing tyrosines at positions 333 and 338 or the same cDNA with codons for amino acids 297-311 deleted. The cDNAs encoding the mutated GHR were generated by polymerase chain reaction as described previously(29, 30, 31) . Cells were screened for GHR expression as described previously(32, 33) . Binding of I-hGH (1 h, 25 °C) to cells expressing GHR1-454, GHR1-294, GHRDeltaP, and GHR1-638(Y333,338F) was 65-100, 200-300, 20-25, and 80-100%, respectively, of binding of I-hGH to cells expressing GHR1-638 (31, and data not shown). The binding affinity for hGH of each of the mutated GHR expressed in CHO cells was similar to that of wild type (6, 29, 31, and data not shown). CHO cells were cultured as described previously(31) .

COS-7 cells were transiently transfected with the cDNA for murine JAK2 or JAK2(K882E) using calcium phosphate precipitation. Briefly, cells were plated in 60-mm dishes and grown to 50% confluence. Expression vector containing the full-length cDNA for JAK2 or JAK2(K882E) (0.1 µg/60-mm plate) was mixed with 0.25 ml of 0.25 M CaCl(2) and 0.25 ml of 50 mM HEPES, 280 mM NaCl, and 1.5 mM Na(2)HPO(4) (pH 7.05). The COS-7 cells were incubated with the DNA mixture for 18 h at 37 °C under 5% CO(2), 95% air. Medium was removed, and Dulbecco's modified Eagle's medium and 10% fetal calf serum were added to the cells for an additional 36 h.

Antibodies

Antibody to SHC (alphaSHC) and Grb2 (alphaGrb2) were from Transduction Laboratories. Antiphosphotyrosine antibody (alphaPY) (4G10) was purchased from UBI. Antibody to JAK2 (alphaJAK2) was a gift of Dr. J. Ihle and was prepared in rabbits against a synthetic peptide corresponding to amino acids 758-776 as described previously(34) . Antibody to GHR (alphaGHBP), kindly provided by Dr. W. R. Baumbach (American Cyanamid, Princeton, NJ), was produced in rabbits using recombinant rat GH binding protein produced in Escherichia coli(35) .

Immunoprecipitation and Western Blotting

As described previously(36) , confluent 3T3-F442A and CHO cells were incubated overnight in the absence of serum. Cells were incubated for the indicated times with hGH at 37 °C in 95% air, 5% CO(2), rinsed with three changes of ice-cold 10 mM sodium phosphate (pH 7.4), 137 mM NaCl, 1 mM Na(3)VO(4) and scraped on ice in lysis buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA, 1 mM Na(3)VO(4), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cell lysates were centrifuged at 12,000 times g for 10 min and the resulting supernatants were incubated on ice for 2 h with the indicated antibody. Immune complexes were collected on protein A-agarose during a 1-h incubation at 8 °C, washed three times with wash buffer (50 mM Tris, pH 7.5, 0.1% Triton X-100, 137 mM NaCl, 2 mM EGTA) and boiled for 5 min in a mixture (80:20) of lysis buffer and 250 mM Tris (pH 6.8), 5% SDS, 10% beta-mercaptoethanol, 40% glycerol. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with the indicated antibody using the ECL detection system(37) . In some experiments, the blots were rinsed in TBS-Tween and Western blotted with a second antibody. All SDS-polyacrylamide gel electrophoresis gels contained prestained molecular weight standards: lysozyme (15,100), beta-lactoglobulin (17,900), carbonic anhydrase (28,250), ovalbumin (43,600), bovine serum albumin (70,800), phosphorylase b (105,000), and myosin (203,000).

Binding Studies Using GST Fusion Proteins and Western Blotting

The procedure followed was the same as described above for immunoprecipitation with two alterations. The cells were scraped in lysis buffer containing 10% glycerol. The supernatants were incubated for 2 h on ice with the indicated fusion protein. Glutathione-agarose was then added for 1 h at 8 °C.


RESULTS

GH Stimulates Tyrosyl Phosphorylation of SHC Proteins

Since SHC involvement in tyrosine kinase signaling pathways appears to require its phosphorylation, we first examined whether GH promotes tyrosyl phosphorylation of SHC proteins. 3T3-F442A cells were incubated with 500 ng/ml (23 nM) hGH for various lengths of time (Fig. 1, lanes A-F) or for 5 min with various concentrations of GH (Fig. 1, lanes G-K). Solubilized proteins were immunoprecipitated with alphaSHC and the presence of phosphorylated tyrosines assessed by Western blotting with alphaPY. Increased tyrosyl phosphorylation of proteins migrating with M(r) values (46,000, 52,000, and 66,000) appropriate for SHC was observed within 1 min following addition of 500 ng/ml GH. Consistent with these bands being SHC proteins, reprobing the blot with alphaSHC revealed 3 bands that co-migrated with the 3 phosphorylated bands (data not shown). The amount of SHC immunoprecipitated did not change with GH, indicating that GH increases the phosphorylation of SHC proteins rather than the amount of SHC (data not shown). A decrease in electrophoretic mobility of phosphorylated 66-kDa SHC was seen after 5-, 15-, and 30-min incubations with 500 ng/ml GH (Fig. 1, lanes D-F), suggesting that GH promotes the phosphorylation of SHC on multiple sites. Increased phosphorylation of the 3 SHC proteins is clearly visible at 50 and 500 ng/ml GH (Fig. 1, lanes J and K). Longer exposures of the autoradiograph in Fig. 1revealed an increase in the phosphorylation of the 66-kDa SHC protein at the two lowest concentrations of hGH tested, 0.5 and 5 ng/ml (data not shown).


Figure 1: GH stimulates tyrosyl phosphorylation of SHC. 3T3-F442A cells were incubated with hGH for the times and concentrations indicated. Cellular proteins were immunoprecipitated with alphaSHC (1:100) and Western blotted with alphaPY (1:7500). The migration of the three antigenically related SHC proteins are indicated on the left.



GH Stimulates Binding of GHR and JAK2 to the SH2 Domain of SHC Proteins

To gain insight into whether the SH2 domain in SHC proteins can bind tyrosyl-phosphorylated GHR and/or JAK2 in a GH-dependent fashion, cell lysates were incubated with the SH2 domain of p46/p52 SHC fused to GST (Fig. 2). Bound proteins were precipitated using glutathione-agarose and Western blotted with alphaPY. A prominent phosphoprotein(s) migrated as a broad band with a M(r) (130,000) appropriate for both GHR and JAK2(1) . Binding of the 130-kDa phosphoprotein(s) to the SHC SH2-GST fusion protein had a time course and GH dose dependence similar to that of SHC phosphorylation (Fig. 1). The 130-kDa phosphoprotein(s) appeared to bind specifically to the SH2 domain of SHC. It did not bind to a GST fusion protein containing Grb2 (data not shown).


Figure 2: GH stimulates binding of 130-kDa phosphoprotein(s) to the SH2 domain of SHC. 3T3-F442A cells were incubated with hGH for the times and concentrations indicated. Cellular proteins were precipitated using SHC SH2-GST fusion protein and bound proteins were Western blotted with alphaPY (1:7500). The migration of the 130-kDa protein(s) is indicated on the left. The migration of the 105-kDa standard is indicated on the right.



To determine whether JAK2 and/or GHR binds the SH2 domain of SHC in a GH-dependent manner, the blot used in Fig. 2was probed with alphaJAK2 instead of alphaPY (Fig. 3, lanes A-K) and duplicate 0 and 5-min samples were Western blotted with alphaGHBP (Fig. 3, lanes L and M). The results shown in Fig. 3confirm that both JAK2 and GHR bind to the SH2 domain of SHC in a GH-dependent manner. Taken together, the results of Fig. 1Fig. 2Fig. 3indicate that the SH2 domain of SHC proteins is capable of binding to the activated tyrosyl-phosphorylated GHRbulletJAK2 complex and that SHC proteins are tyrosyl phosphorylated in response to GH.


Figure 3: GH stimulates binding of JAK2 and GHR to the SH2 domain of SHC. The Western blot used for Fig. 2was probed with alphaJAK2 (1:7500) (lanes A-K). Duplicate 0 and 5-min (500 ng/ml hGH) samples on the same original blot as lanes A-K were probed with alphaGHBP (1:10,000) (lanes L and M). The migration of JAK2 and GHR are indicated.



Tyrosyl-phosphorylated JAK2 Binds to the SH2 Domain of SHC Proteins

Because JAK2 and GHR form a tight complex(1) , the binding of both GHR and JAK2 to the SH2 domain of SHC is consistent with either JAK2 or GHR containing a SHC binding site. To gain insight into whether SHC SH2 domains can associate with JAK2 in the absence of GHR, COS-7 cells were transiently transfected with JAK2 cDNA. COS cells express no detectable GHR and barely detectable levels of JAK2. Furthermore, when expressed in COS-7 cells, at least a portion of JAK2 is constitutively activated and tyrosyl phosphorylated. (^2)Western blotting with alphaJAK2 of an alphaJAK2 immunoprecipitate from the transfected cells (Fig. 4, lane B) was used to determine the migration of JAK2. Western blotting of the same alphaJAK2 immunoprecipitate with alphaPY (Fig. 4, lane A) was used to confirm that at least a portion of the JAK2 expressed in COS-7 cells is tyrosyl phosphorylated. The tyrosyl-phosphorylated JAK2 visualized in Fig. 4, lane A, is believed to be expressed JAK2 rather than endogenous JAK2 because tyrosyl-phosphorylated JAK2 is not detectable in cells transfected with a control plasmid (data not shown).


Figure 4: JAK2 associates with SHC SH2 domain when transfected in COS-7 cells. COS-7 cells were transfected with JAK2 (lanes A, B, and E), JAK2(K882E) (lane C), and control (lane D) cDNAs. Cellular proteins were incubated with alphaJAK2 (1:500, lanes A and B) or SHC SH2-GST fusion protein (lanes C-E) and bound proteins were Western blotted with alphaPY (1:7500, lane A) or alphaJAK2 (1:7500, lanes B-E). The migration of JAK2 and the 105-kDa standard is indicated.



To examine JAK2-SHC association, lysates from the transfected cells were incubated with the SH2 domain of p46/p52 SHC fused to GST. Bound proteins were eluted and Western blotted with alphaJAK2. Fig. 4, lane E, shows that a protein that co-migrates with JAK2 binds to the SH2 domain of SHC. Consistent with this band being JAK2, it was not present when COS-7 cells were transfected with a control plasmid (Fig. 4, lane D). Probing the blot with alphaPY indicated that the JAK2 bound to the SHC SH2 fusion protein is tyrosyl-phosphorylated (data not shown). SHC SH2 domain appeared capable of binding only to tyrosyl-phosphorylated JAK2, since JAK2 association was greatly reduced when the cells were transfected with a cDNA encoding kinase-dead JAK2(K882E) in which the critical lysine in the ATP binding region (34, 38) was mutated to glutamic acid (Fig. 4, lane C). Lysines in the equivalent position in other kinases have been shown to be involved in the phosphotransfer reaction in the ATP binding site. Mutation of this lysine in other kinases to any other amino acid has resulted in the loss of kinase activity(39) . These data suggest that constitutively active JAK2 in the absence of GHR can associate with the SH2 domain of SHC. Tyrosyl phosphorylation appears to be required for this interaction.

Regions of the GHR Required for GH-dependent Tyrosyl Phosphorylation of SHC Proteins

To assess whether interactions with GHR also contribute to GH regulation of the SHC signaling pathway, the ability of SHC proteins to undergo GH-dependent tyrosyl phosphorylation was monitored in CHO cells expressing various GHR mutants (Fig. 5). A GH-dependent increase in tyrosyl phosphorylation of proteins precipitated by alphaSHC and migrating with M(r) values (46,000, 52,000, and 66,000) appropriate for SHC proteins was observed in CHO cells expressing GHR1-638 (wild-type) (Fig. 6, A, lanes B and J, and panel B). A GH-dependent increase in tyrosyl phosphorylation of the 66- and 52-kDa SHC proteins, but not the 46-kDa SHC protein, was observed for GHR1-454 which lacks the C-terminal half of the GHR cytoplasmic domain (Fig. 6, A, lane D, and panel B). No GH-dependent increase in tyrosyl phosphorylation of any SHC protein was detected using CHO cells expressing GHR1-294 which lacks all but 5 amino acids of the cytoplasmic domain of GHR. These data indicate that amino acids present in GHR295-454 are required for GH-dependent phosphorylation of the 66- and 52-kDa SHC proteins and amino acids present in GHR455-638 contribute to GH-dependent phosphorylation of the 46-kDa SHC protein. Amino acids present in GHR295-454 may also be required for a full response of 46-kDa SHC.


Figure 5: Wild-type and mutated GHRs expressed in CHO cells. Denoted are the extracellular domain, the transmembrane domain (hatched area), and the cytoplasmic domain of the various mutated rat liver GHR used in these studies. Tyrosyl residues in the cytoplasmic domain are denoted by ``Y.'' Phenylalanine residues that were substituted for tyrosyl residues are denoted by ``F.''




Figure 6: Ability of mutated GHR to elicit GH-dependent tyrosyl phosphorylation of SHC. A, CHO cells expressing wild type or mutated GHR (see Fig. 5) as indicated were stimulated for 5 min at 37 °C with 500 ng/ml hGH (lanes B, D, F, H, J, and L) or vehicle (lanes A, C, E, G, I, and K). Cellular proteins were immunoprecipitated with alphaSHC (1:100) and Western blotted with alphaPY (1:7500). The migration of the three antigenically related SHC proteins is indicated on the left. Lanes A-H are from one experiment; lanes I-L are from a second experiment. B, the amount of tyrosyl phosphorylation of the different SHC proteins was quantified using either scanning densitometry (BioMed Instruments laser scanning densitometer) or phosphoimaging (Bio-Rad GS-250 Molecular Imager, Molecular Analyst/Macintosh Image Analysis software). Results are expressed as a percentage of control (no GH) values for each cell line. The means ± S.E. are shown. An asterisk denotes that the mean is statistically different at the 95% confidence level from 100% using a one-tailed, paired Student's t test. The values for 52-kDa SHC for CHO1-454 versus CHO1-638 were not statistically different. N = 6, 5, 3, 3, and 4, for GHR1-638, GHR1-454, GHR1-294, GHRDeltaP, and GHR1-638(Y333,338F), respectively.



A proline-rich motif (in GHR, amino acids 298-305) is the only known motif present in the cytoplasmic domain of all members of the cytokine receptor superfamily(40) . Deletion of this motif has been shown to eliminate the ability of GHR to bind JAK2 and of GH to activate JAK2(31, 41) . We therefore examined whether deleting this motif would eliminate GH induction of SHC phosphorylation. In CHO cells expressing GHRDeltaP, which lacks amino acids 297-311, no GH-dependent increase in tyrosyl phosphorylation of SHC proteins was detected (Fig. 6, A, lanes G and H, and panel B). This finding indicates that the proline-rich region of the GHR is required for GH-dependent SHC phosphorylation, suggesting that JAK2 association with GHR and activation is required for SHC phosphorylation.

Four tyrosines are present in the cytoplasmic domain of GHR1-454. Of these, tyrosines 333 and/or 338 is believed to be phosphorylated in response to GH. (^3)To determine if either of these two tyrosines is the major site of SHC protein association with GHR, tyrosines 333 and 338 were mutated to phenylalanines. When GHR1-638(Y333,338F) was expressed in CHO cells, a GH-dependent increase in tyrosyl phosphorylation of the three SHC proteins similar in magnitude to what was observed using cells expressing GHR 1-638 was observed (Fig. 6, A, lane L, and panel B), indicating that tyrosines 333 and 338 are not required for GH-dependent SHC phosphorylation.

GH Stimulates Association of SHC Proteins with Grb2

MAP kinase activation by ligands that bind to receptor tyrosine kinases appears to require the recruitment of Grb2, which has been shown to bind phosphorylated tyrosines in SHC or in the receptor itself(21, 22, 25) . To evaluate whether GH stimulates the association of Grb2 with SHC, 3T3-F442A cells were stimulated with 500 ng/ml hGH for varying lengths of time and for 5 min with varying concentrations of GH (Fig. 7). SHC and associated proteins were immunoprecipitated with alphaSHC and Western blotted with alphaGrb2. A protein recognized by alphaGrb2 in Western blots and migrating with the appropriate size (M(r) 23,000) for Grb2 was precipitated by alphaSHC in a GH-dependent fashion. The time course and dose response of Grb2 association with SHC proteins paralleled the time course and GH dose response of SHC phosphorylation (Fig. 1). They are also consistent with GH-dependent MAP kinase activation occurring via the SHC/Grb2 pathway (4) . In experiments not shown, proteins in 3T3-F442A cell lysates were precipitated with immobilized Grb2-GST fusion protein and Western blotted with alphaGHBP, alphaJAK2, or alphaSHC. Only the Western blot with alphaSHC revealed a GH-dependent increase in association with Grb2. Similarly, when proteins were immunoprecipitated with alphaGH, alphaGHBP, alphaJAK2, or alphaSHC and Western blotted with alphaGrb2, the presence of Grb2 was detected only in the alphaSHC immunoprecipitate (data not shown). These results provide further evidence that GH stimulates Grb2 binding to SHC proteins but not to GHR or JAK2.


Figure 7: GH promotes association of Grb2 with SHC. 3T3-F442A cells were incubated with hGH for the times and concentrations indicated. Cellular proteins were immunoprecipitated with alphaSHC (1:100) and Western blotted with alphaGrb2 (1:250). The migration of Grb2 is indicated.




DISCUSSION

Involvement of SHC and Grb2 Proteins in Signaling by GH

In this paper, we show that GH promotes the binding of GHR and JAK2 to a fusion protein containing the SH2 domain of SHC and that three SHC proteins are rapidly tyrosyl phosphorylated in response to GH. These results suggest that GH promotes the binding of SHC proteins to GHRbulletJAK2 complexes and that these proteins are then phosphorylated on tyrosines (presumably by JAK2), which in turn enables them to bind other SH2-containing proteins. That Grb2 is one of these proteins is suggested by the finding that Grb2 co-precipitates with SHC proteins in a GH-dependent fashion. The findings that Grb2 co-precipitates with SHC proteins, but not with GHR or JAK2, and that SHC, but not GHRbulletJAK2 complexes, binds to Grb2-GST fusion protein indicates that SHC is required for the mobilization of Grb2 by GH. The finding that GH recruits both SHC and Grb2 is consistent with GH activation of MAP kinase involving the SHC/Grb2/son of sevenless/Raf/ras/MEK pathway.

Evidence Supporting the Binding of SHC Proteins to JAK2

Experiments using mutated GHRs expressed in CHO cells implicate the proline-rich motif of GHR in GH-dependent SHC phosphorylation. This motif has previously been implicated in GHRbulletJAK2 association and JAK2 activation by GH(31, 41) . Thus, our results are consistent with a requirement for JAK2 binding and activation prior to SHC tyrosyl phosphorylation. The simplest explanation for this JAK2 dependence is that JAK2 is the kinase that phosphorylates SHC. Additionally, or alternatively, JAK2 may provide phosphorylated tyrosines to which SHC proteins bind.

Several lines of evidence are consistent with SHC proteins binding to phosphorylated tyrosyl residues in JAK2. First, full-length GHR1-638(Y333,338F) is capable of mediating GH-dependent SHC phosphorylation. If SHC proteins bind only to phosphorylated tyrosines and if Tyr and/or Tyr are the only tyrosine is GHR1-454 that is phosphorylated in response to GH, as hypothesized(17) ,^3 then this finding suggests that GHR1-454, which is capable of mediating GH-dependent tyrosyl phosphorylation of SHC proteins, does not itself contain a SHC binding site. Rather, SHC must be binding to a GHR-associated protein (e.g. JAK2). Second, when prepared from GHR-deficient COS cells that express constitutively activated and tyrosyl-phosphorylated murine JAK2 in the absence of GHR, JAK2 is capable of binding the SH2 domain of SHC. Third, based upon amino acid analysis(42, 43, 44) , murine JAK2 contains a number of potential SHC binding sites. The JAK2 sequence YLFV is similar to what is believed to be the highest affinity SHC binding site in epidermal growth factor receptor (YLRV) and the platelet-derived growth factor receptor (YIYV)(17, 43) . Three additional tyrosine-containing motifs in JAK2 (YLKF, YGQL, and YGSL) fit the consensus sequence for SHC binding (Y, hydrophobic, X, hydrophobic) derived from studies of SHC binding to proteins and peptides containing phosphorylated tyrosyl residues(21, 44, 45) .

Possible Binding Sites for SHC in GHR

The results presented here that support the hypothesis that SHC binds to phosphorylated tyrosyl residues in JAK2 do not exclude the possibility that SHC proteins also bind to phosphorylated tyrosyl residues in GHR. Recent evidence suggests that SHC proteins bind multiple tyrosyl phosphorylation sites in both the platelet-derived growth factor and epidermal growth factor receptors(21, 45) . Thus, GHR and JAK2 may both contain SHC binding sites. There are several observations that indirectly support the hypothesis that SHC proteins bind GHR directly. First, the ratio of tyrosyl-phosphorylated JAK2 to tyrosyl-phosphorylated GHR binding to the SH2 domain of SHC more closely resembles the ratio (leq1) observed when GHR and associated proteins are precipitated using antibodies to either GH or GHR than it resembles the ratio (1) observed when JAK2 and its associated proteins are immunoprecipitated using antibody to JAK2(1, 31) . This suggests that SHC proteins have a higher affinity for GHR than for JAK2 and/or that SHC proteins bind JAK2 with higher affinity when JAK2 is in a complex with GHR. Second, GHR1-638 was found to increase tyrosyl phosphorylation of all three SHC proteins, whereas GHR1-454 appeared to increase the phosphorylation of only the 52- and 66-kDa SHC proteins. This suggests that amino acids 455-638 of GHR are required for tyrosyl phosphorylation of 46-kDa SHC protein and that the various forms of SHC may differ in their affinity for specific phosphorylated tyrosyl residues. Third, there is a tyrosine-containing amino acid sequence in the cytoplasmic domain of GHR (YAQV), missing in GHR1-454, that has the required consensus sequence for SHC binding. There are 9 other tyrosines in the cytoplasmic domain of murine GHR whose environment does not match as well the current notion of a SHC binding site, but cannot yet be ruled out as potential SHC binding sites.

While the results of our experiments discussed above support the hypothesis that SHC proteins can bind to JAK2bulletGHR complexes, we have been unable to detect SHC proteins in alphaGHR, alphaGH, or alphaJAK2 immunoprecipitates nor have we been able to detect GHR or JAK2 in alphaSHC immunoprecipitates (data not shown). Potential explanations for this apparent discrepancy include: 1) the antibodies used may be unable to immunoprecipitate SHCbulletGHRbulletJAK2 complexes because their epitopes are either not accessible when the antigens are present in a complex or antibody binding disrupts pre-existing complexes; 2) SHCbulletGHRbulletJAK2 complexes may adventitiously dissociate during the cell lysis and immunoprecipitation steps; 3) the antibodies may be unable to detect in Western blots the potentially small amounts of proteins expected to be co-precipitated. Alternatively, it is possible that while JAK2bulletGHR complexes can bind to the SH2 domain of SHC proteins in vitro, they do not readily do so in intact 3T3-F442A cells where the concentration of SHC proteins is lower and the environment is different. Others have hypothesized the existence of an adaptor protein of M(r) 150,000 that facilitates SHC binding to certain receptors(46) . Arguing against the possibility that SHC association with GHRbulletJAK2 complex occurs indirectly via this or another adaptor protein is our inability to detect any GH-dependent phosphoprotein other than SHC proteins in alphaSHC immunoprecipitates (data not shown). Thus, the simplest interpretation of our findings is that SHC proteins bind directly to GHRbulletJAK2 complexes. However, our data do not exclude the alternative possibility that an additional adaptor protein facilitates binding of SHC to GHRbulletJAK2 complexes.

In conclusion, these and previous results (1) suggest that, in 3T3-F442A cells, GH binds to GHR, promotes association of JAK2 with GHR, and activation of JAK2. The activated JAK2 phosphorylates both itself and GHR. SHC proteins then interact with GHRbulletJAK2 complexes via the SH2 domains of SHC and are phosphorylated by JAK2. The tyrosyl-phosphorylated SHC proteins in turn bind Grb2. This pathway is likely to be an initial response to GH which ultimately leads to MAP kinase activation. Our results suggesting that SHC proteins associate with phosphorylated JAK2 imply that SHC proteins will be recruited in proportion to the amount of JAK2 activated by the numerous ligands capable of activating JAK2 (e.g. GH, prolactin, erythropoietin, interleukin-3, -5, and -6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and interferon-(1, 34, 47, 48, 49, 50) ). However, our evidence suggesting that GHR may also contribute SHC binding sites raises the possibility that the ligands that activate JAK2 may not stimulate SHC phosphorylation simply in proportion to the amount of JAK2 they activate. Those receptors that are more abundant and have higher affinity SHC binding sites would be expected to recruit SHC proteins to a greater extent than those that are less abundant and have lower affinity SHC binding sites. In this regard, it has been reported that of the cytokines known to activate JAK2, interleukins-3 and -5, erythropoietin, and granulocyte-macrophage colony-stimulating factor stimulate tyrosyl phosphorylation of SHC proteins(23, 51, 52) . These cytokines as well as interleukin-6, leukemia inhibitory factor, and oncostatin M, have been reported to activate MAP kinases(53, 54, 55) . It will be interesting to determine for these cytokines whether SHC proteins interact with receptor, JAK2, or some other protein.


FOOTNOTES

*
This work was supported in part by Research Grant ROI-DK34171 from the National Institutes of Health (to C. C.-S.). Computer studies were supported in part by the General Clinical Research Center at The University of Michigan, funded by Grant M01 RR00042 from The National Center for Research Resources, National Institutes of Health, United States Public Health Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of postdoctoral fellowships from the National Institutes of Health (5T32-DK07245) and the Arthritis Foundation.

To whom correspondence should be addressed: Dept. of Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622. Fax: 313-936-8813.

(^1)
The abbreviations used are: GH, growth hormone; hGH, human growth hormone; GHR, growth hormone receptor; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase; SH, Src homology; Grb2, growth factor receptor bound 2; CHO, Chinese hamster ovary; GST, glutathione S-transferase.

(^2)
W. H. Huo, L. S. Argetsinger, G. S. Campbell, and C. Carter-Su, manuscript in preparation.

(^3)
J. VanderKuur, X. Wang, N. Billestrup, L. Zhang, and C. Carter-Su, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. L. S. Argetsinger and G. S. Campbell for their helpful comments and review of the manuscript and D. Kim and L. Zhang for help with the cell culture. We are grateful to Drs. J. Ihle and B. Witthuhn for providing the cDNAs for JAK2 and JAK2(K882E); Drs. S. Decker and A. Saltiel for providing Grb2 and SHC SH2-GST fusion proteins; and Dr. W. R. Baumbach for antibody to GHR.


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