Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active.
Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active.
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DOI：
10.1016/j.cellsig.2010.10.024
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发表时间：
2011-02
影响因子：
4.8
通讯作者：
Clerk A
中科院分区：
文献类型：
--
作者：
Sugden PH;Markou T;Fuller SJ;Tham el L;Molkentin JD;Paterson HF;Clerk A
ERK 1和ERK 2（ERK 1/2）是调节细胞分裂，生长和存活的核心。通过Thr-Glu-Tyr活化环中Thr-和Tyr-残基的磷酸化活化。教条是双重磷酸化的ERK 1/2构成完整细胞中的主要活性。我们以前表明，在新生大鼠心肌细胞，内皮素-1和佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）强大而迅速（最大约5分钟）激活ERK 12。在这里，我们发现双重磷酸化的ERK 1/2在内皮素-1刺激后迅速（< 2分钟）出现在细胞核中。我们使用MonoQ FPLC表征了暴露于内皮素-1或PMA的心肌细胞中的活性ERK 1/2种类。出乎意料的是，使用体外激酶测定，ERK 1的两个峰和ERK 2活性的两个峰被解析。这些中的每一个代表双磷酸化物质。另外两个代表仅在Thr-残基上磷酸化的ERK 1或ERK 2的活性。在内皮素-1或PMA刺激后，单磷酸苏糖基ERK 1/2最多占总ERK 1/2活性的约30%，估计其kcat值最低为双磷酸化物质的约30%。与双磷酸化ERK 1/2相比，单磷酸苏糖基ERK 1/2的出现迅速但延迟。在研究的10种激动剂中，内皮素-1和PMA在ERK 1/2激活和刺激单磷酰和双磷酸化ERK 1/2的出现方面最有效。因此，酶促活性的单磷苏氨酰ERK 1/2形成内源性的ERK 1/2级联激活后，我们认为，单磷苏氨酰ERK 1/2产生的蛋白酪氨酸磷酸酶介导的去磷酸化的双重磷酸化的ERK 1/2。
ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~ 5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~ 30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their kcat values were estimated to be minimally ~ 30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.
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