VanderKuur J, Allevato G, Billestrup N, Norstedt G, Carter-Su C
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622, USA.
J Biol Chem. 1995 Mar 31;270(13):7587-93. doi: 10.1074/jbc.270.13.7587.
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.
生长激素（GH）已被证明可刺激称为细胞外信号调节激酶（ERK）1和2的丝裂原活化蛋白（MAP）激酶。ERK1和2被酪氨酸激酶激活的一条途径涉及含Src同源（SH）-2结构域的蛋白质SHC和Grb2。为深入了解将生长激素受体（GHR）与MAP激酶激活相偶联的途径以及可能与GHR及其相关酪氨酸激酶JAK2相互作用的信号分子，我们研究了SHC和Grb2蛋白是否作为GH的信号分子。结果显示，人GH可促进3T3-F442A成纤维细胞中66 kDa、52 kDa和46 kDa的SHC蛋白发生快速酪氨酰磷酸化。GH还促进GHR和JAK2与融合至谷胱甘肽S-转移酶（GST）的46/52 kDa SHC蛋白的SH2结构域结合。用鼠JAK2 cDNA瞬时转染的COS-7细胞中组成性磷酸化的JAK2与SHC SH2-GST融合蛋白结合，表明在无GHR的情况下，SHC SH2结构域可结合酪氨酰磷酸化的JAK2。使用表达突变大鼠GHR的中国仓鼠卵巢细胞研究了GH依赖性SHC酪氨酰磷酸化所需的GHR区域。在表达GHR1-638和GHR1-638（Y333,338F）的细胞中，GH刺激所有3种SHC蛋白的磷酸化，而在表达GHR1-454的细胞中，GH仅刺激66 kDa和52 kDa的SHC蛋白磷酸化。GH对表达GHR1-294或GHRδP的细胞中的SHC磷酸化无影响，后者缺乏含GH激活JAK2所需富含脯氨酸基序的297 - 311位氨基酸。与SHC不同，Grb2似乎不直接与GHR或JAK2相互作用。然而，Grb2被证明以GH依赖性方式迅速与SHC蛋白结合。这些发现表明，GH刺激：1）SHC蛋白通过SHC-SH2结构域与JAK2-GHR复合物结合；2）SHC蛋白的酪氨酰磷酸化；3）随后Grb2与SHC蛋白结合。这些事件可能是GH激活MAP激酶以及可能对GH的其他反应中的早期事件。
