Activation of focal adhesion kinase by protein kinase Cε in neonatal rat ventricular myocytes
Activation of focal adhesion kinase by protein kinase Cε in neonatal rat ventricular myocytes
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DOI：
10.1152/ajpheart.00016.2003
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发表时间：
2003-10-01
影响因子：
4.8
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
局灶黏附激酶（FAK）是一种非受体蛋白酪氨酸激酶，在内皮素（ET）诱导的心肌细胞肥大过程中，对心肌细胞存活和肌体组装都至关重要。et诱导的FAK激活需要上游激活一个或多个蛋白激酶C （PKC）同工酶。因此，我们利用复制缺陷腺病毒（Adv）过表达PKC的组成活性（ca）和显性阴性（dn）突变体，研究了哪些PKC同工酶是FAK激活所必需的，以及哪些下游信号成分参与其中。通过Western blot分析，FAK活化的特异性抗体在Y397位点自磷酸化（Y397pFAK）。ET （10 nmol/l, 2 ~ 30 min）可使FAK产生时间依赖性的活化，而chelerythrine （5 mumol/l，预处理1 h）可抑制FAK的活化。与编码β -半乳糖苷酶的Adv相比，AdvcaPKCepsilon激活了FAK，而Adv- capkcdelta没有激活FAK。相反，Adv-dnPKCepsilon抑制et诱导的FAK激活。Y-27632 （10 μ mol/l，预处理1小时）是一种rho相关的含卷曲卷曲蛋白激酶（ROCK）抑制剂，可阻止ET-和capkcepsilon诱导的FAK活化以及cofilin磷酸化。细胞松弛素D预处理（1 μ mol/l，预处理1 h）也能抑制ET诱导的Y397pFAK和cofilin磷酸化以及capkcepsilon诱导的Y397pFAK。然而，两种抑制剂均未干扰ET诱导的ERK1/2激活。最后，PP2 （50 μ mol/l，预处理1小时），一种高选择性Src抑制剂，不会改变基础或et诱导的Y397pFAK。然而，PP2确实降低了基础磷酸化和ET诱导的FAK上其他位点的磷酸化，即Y576、Y577、Y861和Y925。我们得出结论，et诱导的下游Y397pFAK信号转导通路部分依赖于PKCepsilon、ROCK、cofilin和组装的肌动蛋白丝，而不是ERK1/2或Src。
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET ( 10 nmol/l; 2 - 30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine ( 5 mumol/l; 1 h pretreatment). AdvcaPKCepsilon, but not Adv-caPKCdelta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKCepsilon inhibited ET-induced FAK activation. Y-27632 ( 10 mumol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKCepsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 mumol/l, 1 h pretreatment) also inhibited ET- induced Y397pFAK and cofilin phosphorylation and caPKCepsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET- induced ERK1/2 activation. Finally, PP2 (50 mumol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET- induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKCepsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.