GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes
GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes
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DOI：
10.1161/01.res.0000023201.41774.ea
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发表时间：
2002-06-28
影响因子：
20.1
通讯作者：
Samarel, AM
中科院分区：
文献类型：
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作者：
Heidkamp, MC;Bayer, AL;Samarel, AM
粘着斑激酶 (FAK) 是一种参与粘附依赖性信号转导的非受体蛋白酪氨酸激酶。 FAK 在培养的新生大鼠心室肌细胞 (NRVM) 中高度表达，并响应细胞粘附、拉伸和生长因子刺激而发生酪氨酸自磷酸化。我们之前表明，通过腺病毒介导的 FRNK（FAK 自主表达的 C 端结构域）过表达来抑制 FAK 磷酸化可防止内皮素-1 (ET) 诱导的 NRVM 肥大。这些研究提出的一个问题是 FRNK 是否定位于粘着斑并从下游信号传导所需的位点取代了 FAK。因此，我们构建了编码 GFP-FRNK 融合蛋白 (Adv-GFP-FRNK) 的复制缺陷型腺病毒，并检查了其对 NRVM 细胞结构和信号传导的影响。未感染的 NRVM 含有少量内源性 FRNK。感染 Adv-GFP-FRNK 的 NRVM 表达大量的 66-/68-kDa 蛋白，该蛋白定位于肋节和粘着斑。 GFP-FRNK 过表达抑制基础和 ET 诱导的 FAK 磷酸化，还抑制 ET 诱导的 PYK2（非受体蛋白酪氨酸激酶 FAK 家族的另一个成员）的磷酸化。相反，GFP-FRNK 过表达并不能阻止 ET 诱导的 ERK、JNK 或 p70S6K 磷酸化。此外，GFP-FRNK 导致粘着斑中可检测到的 FAK 和桩蛋白丢失，同时伴随着总桩蛋白水平降低，最终导致细胞脱离和凋亡。我们得出的结论是，FRNK 通过取代粘着斑中的 FAK 并干扰细胞生存所必需的 NRVM 的锚定，作为粘附依赖性信号传导的显性失活抑制剂，这一过程称为失巢凋亡。
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis.