Hannan Katherine M, Thomas George, Pearson Richard B
Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Locked Bag #1, A'Beckett Street, Melbourne, Victoria 8006, Australia.
Biochem J. 2003 Mar 1;370(Pt 2):469-77. doi: 10.1042/BJ20021709.
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 (398-502) 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 auto-inhibitory 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.
丝裂原刺激的蛋白激酶p70核糖体蛋白S6激酶1（S6K1）是细胞生长和增殖调控中的关键酶。S6K1的激活需要一系列复杂、有序的构象变化和磷酸化反应。虽然连续多位点磷酸化的作用已得到广泛详细的描述，但启动这一级联反应所需的引发步骤的特征仍不清楚。在本研究中，我们首次表明这一引发过程依赖于钙。钙对S6K1的依赖性调节并非特异性靶向关键调节磷酸化位点苏氨酸-229和苏氨酸-389；相反，钙螯合导致S6K1磷酸化的全面抑制。将自抑制域和疏水域中单个磷酸化位点突变为酸性残基（以模拟磷酸化）产生的激酶对钙螯合仍敏感，而联合突变则减轻了对钙的需求。此外，C末端残基（398 - 502）的缺失也使激酶对钙不敏感。我们推测，最初的钙依赖性过程是为了释放S6K1的C末端和N末端之间的抑制性相互作用，从而允许这些关键结构域的磷酸化。只有通过模拟自抑制域和疏水域磷酸化的突变才能克服对这一引发步骤的需求。我们进一步提出，引发事件涉及形成一种钙依赖性蛋白复合物，该复合物释放N末端和C末端之间的相互作用。然后，S6K1可被靶向已知调节磷酸化位点的激酶激活。与这一假设一致，血清刺激S6K1活性与其掺入钙依赖性高分子量复合物有关。
