Crute B E, Seefeld K, Gamble J, Kemp B E, Witters L A
Endocrine-Metabolism Division, Departments of Medicine and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3833, USA.
J Biol Chem. 1998 Dec 25;273(52):35347-54. doi: 10.1074/jbc.273.52.35347.
The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic alpha subunit and noncatalytic beta and gamma subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of Saccharomyces cerevisiae. In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the alpha1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits and dramatically increases protein expression. The full-length wild-type alpha1 subunit is only minimally active in the absence of co-expressed beta/gamma, and alpha1(1-392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. alpha-1(1-312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated alpha subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the alpha1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.
AMP激活的蛋白激酶是一种异源三聚体酶，在细胞适应营养饥饿、缺氧、ATP利用增加或热休克等应激过程中起重要作用。这种哺乳动物酶由一个催化性α亚基和非催化性β和γ亚基组成，是一个更大的蛋白激酶家族的成员，该家族包括酿酒酵母的SNF1激酶。在本研究中，我们通过截短和定点诱变鉴定了α1催化亚基的几个功能结构域，这些结构域调节其活性、亚基缔合和蛋白质周转。将548个氨基酸（aa）的野生型α1蛋白C末端截短至312或392个氨基酸可消除β/γ亚基的结合，并显著增加蛋白质表达。在没有共表达的β/γ的情况下，全长野生型α1亚基的活性极低，α1(1-392)同样活性很小。然而，进一步截短至312个氨基酸与酶比活性的大幅增加相关，从而揭示了313至392个氨基酸之间的自抑制序列。α-1(1-312)仍需要激活环苏氨酸-172的磷酸化才能发挥酶活性，但现在不依赖于变构激活剂AMP。通过脉冲追踪分析，截短的α亚基cDNA瞬时转染后蛋白质表达水平的增加是由于酶周转的减少。综上所述，这些数据表明AMP激活的蛋白激酶的α1亚基包含几个决定酶活性和稳定性的特征。一种不需要非催化亚基参与的组成型活性激酶形式为探索AMP激活的蛋白激酶的功能提供了一种独特的试剂。
