Activation of S6K1 (p70 ribosomal protein S6 kinase 1) requires an initial calcium-dependent priming event involving formation of a high-molecular-mass signalling complex
Activation of S6K1 (p70 ribosomal protein S6 kinase 1) requires an initial calcium-dependent priming event involving formation of a high-molecular-mass signalling complex
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DOI：
10.1042/bj20021709
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发表时间：
2003-03-01
影响因子：
4.1
通讯作者：
Pearson, RB
中科院分区：
文献类型：
--
作者：
Hannan, KM;Thomas, G;Pearson, RB
丝裂原刺激蛋白激酶p70核糖体蛋白S6激酶1(S6K1)是调节细胞生长和增殖的关键酶。S6K1的激活需要一系列复杂、有序的构象变化和磷酸化反应。虽然顺序的、多位点的磷酸化的作用已经被广泛地详细描述，但启动这一级联反应所需的启动步骤的特征仍然难以捉摸。在目前的研究中，我们第一次证明了这种启动过程依赖于钙。S6K1的钙依赖性调节并不针对关键的调节磷酸化位点Thr-229和Thr-389；相反，钙离子螯合导致S6K1磷酸化的整体抑制。将自身抑制和疏水结构域中的单个磷酸化位点突变为酸性残基(以模拟磷酸化)可产生对钙离子螯合保持敏感的激酶，而组合突变可缓解对钙的需求。此外，C末端残基(398502)的缺失也使该激酶对钙不敏感。我们假设，需要最初的钙依赖过程来释放S6K1的C-端和N-端之间的抑制相互作用，从而允许这些关键结构域的磷酸化。只有通过模拟自抑制和疏水结构域的磷酸化的突变才能克服这一启动步骤的要求。我们进一步认为，启动事件涉及形成一个钙依赖的蛋白质复合体，释放N-末端和C-末端之间的相互作用。然后，S6K1就可以被以已知调节磷酸化位点为靶点的激酶激活。与这一假设一致的是，血清对S6K1活性的刺激与其掺入钙依赖的高分子复合体有关。
The mitogen-stimulated protein kinase p70 ribosomal protein S6 kinase 1 (S6K1) is a key enzyme in the regulation of cell growth and proliferation. Activation of S6K1 requires a complex, ordered series of conformational changes and phosphorylation reactions. While the role of sequential, multi-site phosphorylation has been extensively detailed, characterization of the priming step required to initiate this cascade has remained elusive. In the present study we show for the first time that this priming process is dependent on calcium. Calcium-dependent regulation of S6K1 did not specifically target Thr-229 and Thr-389, the key regulatory phosphorylation sites; rather, calcium chelation resulted in a global inhibition of S6K1 phosphorylation. Mutation of individual phosphorylation sites in the auto-inhibitory and hydrophobic domains to acidic residues (to mimic phosphorylation) yields a kinase that remains sensitive to calcium chelation, while the combined mutations alleviate the requirement for calcium. Furthermore, deletion of the C-terminal residues (398502) also renders the kinase insensitive to calcium. We hypothesize that the initial calcium-dependent process is required to release an inhibitory interaction between the C- and N-termini of S6K1, thus allowing phosphorylation of these key domains. The requirement for this priming step can only be overcome by mutations mimicking the phosphorylation of both the autoinhibitory and hydrophobic domains. We further propose that the priming event involves formation of a calcium-dependent protein complex that releases the interaction between the N- and C-termini. S6K1 is then accessible for activation by the kinases that target the known regulatory phosphorylation sites. Consistent with this hypothesis, serum stimulation of S6K1 activity is associated with its incorporation into a calcium-dependent high-molecular-mass complex.