Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway
Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway
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DOI：
10.1152/ajpendo.00541.2012
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发表时间：
2013-07-01
影响因子：
5.1
通讯作者：
Atherton, Philip J.
中科院分区：
文献类型：
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作者：
Crossland, Hannah;Kazi, Abid A.;Atherton, Philip J.
粘着斑激酶（FAK）是一种附着复合物蛋白，通过目前尚不清楚的机制与肌肉质量的调节有关。我们测试了FAK是否在肌肉肥大的功能上是重要的，假设FAK敲低（FAK-KD）会阻碍与营养刺激相关的细胞生长。使用慢病毒转染技术产生携带FAK靶向（FAK-KD）或乱序（SCR）shRNA的C2 C12骨骼肌细胞。将FAK-KD和SCR肌管与IGF-I（10 ng/ml）一起孵育24小时，并将另外的SCR细胞（+/- IGF-1）与FAK激酶抑制剂一起孵育，然后测定细胞生长。肌肉蛋白合成（MPS）和假定的FAK信号传导机制（免疫印迹和免疫共沉淀）进行了评估。24 h后，FAK-KD细胞中IGF-I诱导的肌管宽度（+41 +/- 7% vs.非IGF-I处理）和总蛋白（+44 +/- 6%）增加减弱，而4 h后FAK-KD vs. SCR中MPS受到抑制。这些钝化的反应与减弱的IGF-I诱导的FAK Tyr（397）磷酸化和显著抑制FAK shRNA细胞中结节性硬化症复合体2（TSC 2）和关键下游mTOR信号传导（核糖体S6激酶，eIF 4F组装）的磷酸化相关（所有P < 0.05，与IGF-I处理的SCR细胞相比）。然而，FAK与TSC 2或其磷酸酶Shp-2的结合不受IGF-I或细胞表型的影响。最后，FAK-KD介导的细胞生长抑制通过直接抑制SCR细胞中的FAK激酶活性来重现。我们得出结论，FAK是IGF-I诱导的肌肉肥大所必需的，通过TSC 2/mTOR/S6 K1依赖性途径通过需要FAK的激酶活性但不改变FAK-TSC 2或FAK-Shp-2结合的方式进行信号传导。
Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C2C12 skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (+/- IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 +/- 7% vs. non-IGF-I-treated) and total protein (+44 +/- 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr(397) phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.