Elbing Karin, McCartney Rhonda R, Schmidt Martin C
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Biochem J. 2006 Feb 1;393(Pt 3):797-805. doi: 10.1042/BJ20051213.
Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.
蛋白激酶Snf1/AMPK家族的成员由不同的上游激酶激活,这些上游激酶使Snf1/AMPK激活环中的一个保守苏氨酸残基磷酸化。最近,Snf1和AMPK激活激酶的身份已被确定。在此,我们描述了酿酒酵母中三种Snf1激活激酶的纯化和特性。通过肽质量指纹图谱确定了与Snf1激活激酶相关的蛋白质的身份。这些激酶Sak1、Tos3和Elm2似乎不需要其他亚基的存在就能发挥活性。Sak1和Snf1在尺寸排阻色谱中共同纯化并共同洗脱,表明这两种蛋白质形成了一个稳定的复合物。Snf1激活激酶在体外以高特异性磷酸化Snf1的激活环苏氨酸,并且在没有Snf1异源三聚体的β和γ亚基的情况下也能这样做。最后,我们表明从细菌中分离出来的作为GST融合蛋白的Snf1激酶结构域在体外可以被激活,并且在没有其β和γ亚基的情况下表现出底物特异性。