Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation.
Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation.
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DOI：
10.1210/endo.141.9.7673
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发表时间：
2000-09
期刊：
影响因子：
4.8
通讯作者：
L. Liang;J. Jiang;S. Frank
中科院分区：
文献类型：
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作者：
L. Liang;J. Jiang;S. Frank
生长激素与细胞表面生长激素受体(GHR)的相互作用导致GHR相关的酪氨酸激酶JAK2的激活，从而触发包括STAT、Ras/Raf/MEK1/MAP激酶和胰岛素受体底物-1(IRS-1)/PI3Kinase在内的信号级联反应。我们先前的研究表明，与仅表达GHR的细胞(32D-rbGHR)相比，稳定表达兔GHR和大鼠IRS-1(32D-rbGHR-IRS-1)的IRS和GHR缺陷的32D细胞表现出明显的GH诱导的增殖和MAP激酶(ERK1和ERK2)的激活。我们现在研究胰岛素样生长因子-1增强生长激素诱导的MAP激酶激活的生化机制(S)。时程实验显示，生长激素诱导的ERK1和ERK2在32D-rbGHR和32D-rbGHR-IRS-1细胞中都有类似的瞬时(15分钟最大)激活，但与我们之前的发现一致，在含有IRS-1的细胞中发现了更大的激活。在这两种细胞中，MEK1抑制剂PD98059可显著抑制GH诱导的MAPK激活，而PKC抑制剂GF109203X则不能。有趣的是，在32D-rbGHR和32D-rbGHR-IRS-1细胞中，用PI3K抑制剂Wortmannin(EC50约10 nM)预处理可显著降低GH诱导的MAPK活性。在依赖IRS-1增强和IRS-1非依赖wortmannin敏感性的两个细胞中，也观察到GH诱导Akt和MEK1的激活(使用状态特异性抗体印迹)，尽管两个细胞之间GHR、JAK2、SHP-2、P85、Akt、RAS、Raf-1、MEK1、ERK1或ERK2的丰度没有差异。另一种PI3K抑制剂LY294002(50微米)基本上抑制(约72%)GH诱导的32D-rbGHR-IRS-1细胞的MAP激酶激活，但仅轻微地(且统计上不显著)抑制GH诱导的32D-rbGHR细胞的MAP激酶激活。由于相同浓度的LY294002可完全抑制GH诱导的Akt激活，这些结果表明IRS-1非依赖和GH依赖的MAP激酶激活对Wortmannin的敏感性可能反映了除PI3K外，另一个Wortmannin敏感靶点(PI3K)在介导GH诱导的MAP激酶激活中的活性。值得注意的是，GH诱导的STAT5酪氨酸磷酸化，不同于Akt或MAPK的激活，在不同的细胞之间没有区别。最后，虽然GH促进了两个细胞中激活的RAS的积累，但表达IRS-1的细胞的基础RAS水平和GH诱导的激活RAS水平都高于32D-rbGHR细胞。这些结果表明，虽然生长激素诱导STAT5酪氨酸磷酸化，并激活Ras/Raf/MEK1/MAPK和PI3K通路，但IRS-1的表达对后两者的促进作用强于前者。
Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.