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 GHR delta P, 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 JAK2.GHR 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.
The relationship between steroid structure, estrogen receptor (ER) binding affinity, nuclear binding of the ER complex, and induction of progesterone receptor (PgR) have been examined. The level of ER in membrane-free homogenates of MCF-7 cells was found to be 10.0 +/- 0.5 fmol/micrograms of DNA by utilizing an enzyme immunoassay (EIA). However, only 2.5 +/- 0.2 fmol of ER complex/micrograms of DNA was bound by nuclei during maximal stimulation of PgR synthesis (2.9 +/- 0.2 fmol of PgR/micrograms of DNA; measured by EIA) following a pulse with 10(-10) M E2. Except at micromolar concentrations, estratriene was an ineffective estrogen. The addition of a hydroxyl group to either position 3 or position 17 beta of estratriene yielded ligands which were capable of causing nuclear binding and processing of ER as well as PgR induction. D-ring regioisomers of estradiol (E2) had lower affinity for receptor than E2. However, receptor complexed with these estrogens was fully capable of binding to nuclear material, undergoing processing, and inducing PgR. On the other hand, A-ring regioisomers of E2 displayed significant differences in their ability to mediate nuclear binding of receptor complex and induction of PgR. Although 1-hydroxyestratrien-17 beta-ol was weakly bound by ER, this dihydroxyestrogen was capable of bringing about nuclear binding and processing of ER and the stimulation of PgR synthesis. In contrast, 2- and 4-hydroxyestratrien-17 beta-ol, which caused extensive nuclear binding of ER (5-7 fmol/micrograms of DNA), were incapable of significant PgR induction.(ABSTRACT TRUNCATED AT 250 WORDS)
We have shown previously that GH stimulates the mitogen-activated protein (MAP) kinases designated ERKs (extracellular signal-regulated kinases) 1 and 2. To examine pathways coupling GH receptor (GHR) to MAP kinase activation, we have determined the effects of GH on SHC-growth factor receptor bound 2-son of Sevenless (SHC-Grb2-SOS) association and activation of Ras, Raf, and MAP-ERK kinase (MEK). GH promoted the rapid, transient association of SHC with the Grb2-SOS complex, which correlated with the time course of Ras, Raf, and MEK activation. Despite the continuous presence of GH, these activation events were transient with Ras, Raf, and MEK returning to near basal activity by 15 or 30 min. The inactivation of Ras, Raf, and MEK directly correlated with the serine/threonine phosphorylation of SOS and dissociation of SOS from Grb2 but not Grb2 from tyrosine-phosphorylated SHC. Phosphorylation was blocked by the MEK inhibitor, PD98059. Based upon the established functions of the MAP kinase pathway, these data indicate that GH stimulation results in the assembly of a SHC-Grb2-SOS complex that serves to activate Ras and thereby engage the Raf-MEK-ERK pathway. Activation of this pathway generates a feedback kinase cascade that phosphorylates SOS resulting in the dissociation of SHC-Grb2 complexes from SOS, thereby causing a more rapid termination of the signaling pathway than would result from SHC dephosphorylation.
Cholecystokinin (CCK) has recently been shown to activate the mitogen-activated protein kinase (MAPK) cascade (Ras-Raf-MAPK kinase-MAPK) in pancreatic acini. The mechanism by which the Gq protein-coupled CCK receptor activates Ras, however, is currently unknown. Growth factor receptors are known to activate Ras by means of adaptor proteins that bind to phosphotyrosine domains. We therefore compared the effects of CCK and epidermal growth factor (EGF) on Tyr phosphorylation of the adaptor proteins Shc and its association with Grb2 and the guanine nucleotide exchange factor SOS. Three major isoforms of Shc (p46, p52, p66) were detected in isolated rat pancreatic acini with p52 Shc being the predominant form. CCK and EGF increased tyrosyl phosphorylation of Shc (251 and 337% of control, respectively). CCK-stimulated tyrosyl phosphorylation of Shc as well as Shc-Grb2 complex formation was significant at 2.5 min, maximal at 5 min, and persisted for at least 30 min. Finally, SOS was found to be associated with Grb2 as assessed by probing of anti-Grb2 immunoprecipitates with anti-SOS. Since MAPK in pancreatic acini is activated via protein kinase C (PKC), we studied the effect of phorbol esters on Shc phosphorylation and found 12-O-tetradecanoylphorbol-13-acetate to be as potent as CCK. Furthermore, GF-109203X, a PKC inhibitor, abolished the effect of 12-O-tetradecanoylphorbol-13-acetate and also the effect of CCK but not the effect of EGF on Shc tyrosyl phosphorylation. CCK-induced tyrosyl phosphorylation of Shc was found to be phosphatidylinositol 3-kinase-independent, and CCK did not cause EGF receptor activation. These results suggest that formation of an Shc-Grb2-SOS complex via a PKC-dependent mechanism may provide the link between Gq protein-coupled CCK receptor stimulation and Ras activation in these cells.
These experiments were designed to examine the effect of structural modifications to the estradiol-17 beta (E2) molecule on the estrogen response element (ERE) dependent activation of the thymidine kinase (tk) promoter. Estrogen receptor (ER) positive MCF-7 cells were transfected with plasmids containing one or two vitellogenin EREs inserted upstream of the tk promoter in p(-37)tk. Transient expression of the CAT gene in these constructs was measured after cells had been maintained for 36-42 h in the presence of E2 or an E2 analogue. E2 induced CAT expression at levels as low as 10(-13) M, with maximum induction at 10(-11) M. CAT activity decreased at higher concentrations of E2. Estratriene, which has low affinity for ER, was active only at micromolar concentrations. 3-Hydroxyestratriene displayed maximal activity at 10(-9) M, with higher levels being less active. Still higher concentrations (10(-7) M) of estratrien-17 beta-ol were required to induce maximum CAT activity. All positional and conformational alterations in the D-ring hydroxyl group of E2 yielded active ligands. Movement of the phenolic hydroxyl group of E2 to other positions on the A-ring produced dihydroxyestrogens with varied capacities to activate CAT (2-hydroxyestratrien-17 beta-ol produced maximum CAT activation at 10(-11) M; 1-hydroxyestratrien-17 beta-ol required a 10(-8) M concentration for maximum activity; 4-hydroxyestratrien-17 beta-ol gave maximum CAT activation at 10(-6) M). Only those androstanediols or 5-androstenediols with a 3 beta-hydroxyl group were capable of activating CAT expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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