Functional Domains of the α1 Catalytic Subunit of the AMP-activated Protein Kinase

Abstract

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 α subunit and noncatalytic β and γ subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of <i>Saccharomyces cerevisiae.</i> In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the α1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type α1 protein to aa 312 or 392 abolishes the binding of the β/γ subunits and dramatically increases protein expression. The full-length wild-type α1 subunit is only minimally active in the absence of co-expressed β/γ, and α1(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. α-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 α subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the α1 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.