Lizcano Jose M, Deak Maria, Morrice Nick, Kieloch Agnieszka, Hastie C James, Dong Liying, Schutkowski Mike, Reimer Ulf, Alessi Dario R
Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, United Kingdom.
J Biol Chem. 2002 Aug 2;277(31):27839-49. doi: 10.1074/jbc.M202042200. Epub 2002 May 22.
The AGC family of protein kinases, which includes isoforms of protein kinase B (also known as Akt), ribosomal S6 protein kinase (S6K), and serum- and glucocorticoid-induced protein kinase (SGK) are activated in response to many extracellular signals and play key roles in regulating diverse cellular processes. They are activated by the phosphorylation of the T loop of their kinase domain by the 3-phosphoinositide-dependent protein kinase-1 and by phosphorylation of a residue located C-terminal to the kinase domain in a region termed the hydrophobic motif. Recent work has implicated the NIMA (never in mitosis, gene A)-related kinase-6 (NEK6) as the enzyme that phosphorylates the hydrophobic motif of S6K1 in vivo. Here we demonstrate that in addition to phosphorylating S6K1 and SGK1 at their hydrophobic motif, NEK6 also phosphorylates S6K1 at two other sites and phosphorylates SGK1 at one other site in vitro. Employing the Jerini pepSTAR method in combination with kinetic analysis of phosphorylation of variant peptides, we establish the key substrate specificity determinants for NEK6. Our analysis indicates that NEK6 has a strong preference for Leu 3 residues N-terminal to the site of phosphorylation. Its mutation to either Ile or Val severely reduced the efficacy with which NEK6-phosphorylated peptide substrates, and moreover, mutation of the equivalent Leu residue in S6K1 or SGK1 prevented phosphorylation of their hydrophobic motifs by NEK6 in vitro. However, these mutants of S6K1 or SGK1 still became phosphorylated at their hydrophobic motif following insulin-like growth factor-1 stimulation of transfected 293 cells. This study provides the first description of the basis for the substrate specificity of NEK6 and indicates that NEK6 is unlikely to be responsible for the IGF1-induced phosphorylation of the hydrophobic motif of S6K, SGK, and protein kinase B isoforms in vivo.
蛋白激酶AGC家族包括蛋白激酶B（也称为Akt）、核糖体S6蛋白激酶（S6K）和血清及糖皮质激素诱导蛋白激酶（SGK）的同工型，它们响应多种细胞外信号而被激活，并在调节多种细胞过程中发挥关键作用。它们通过3-磷酸肌醇依赖性蛋白激酶-1对其激酶结构域的T环进行磷酸化以及在激酶结构域C末端一个称为疏水基序的区域中的一个残基进行磷酸化而被激活。最近的研究表明，NIMA（有丝分裂中从不出现，基因A）相关激酶6（NEK6）是体内磷酸化S6K1疏水基序的酶。在此我们证明，除了在S6K1和SGK1的疏水基序处进行磷酸化外，NEK6在体外还在另外两个位点对S6K1进行磷酸化，并在另一个位点对SGK1进行磷酸化。采用耶里尼pepSTAR方法并结合对变异肽磷酸化的动力学分析，我们确定了NEK6的关键底物特异性决定因素。我们的分析表明，NEK6对磷酸化位点N端的3个亮氨酸残基有强烈偏好。将其突变为异亮氨酸或缬氨酸会严重降低NEK6对磷酸化肽底物的作用效率，此外，S6K1或SGK1中同等亮氨酸残基的突变会阻止NEK6在体外对其疏水基序进行磷酸化。然而，在转染的293细胞经胰岛素样生长因子-1刺激后，S6K1或SGK1的这些突变体在其疏水基序处仍会发生磷酸化。本研究首次描述了NEK6底物特异性的基础，并表明NEK6不太可能是体内胰岛素样生长因子-1诱导的S6K、SGK和蛋白激酶B同工型疏水基序磷酸化的原因。
