The AMP-activated protein kinase is a heterotrimeric enzyme, important in c
ellular adaptation to the stress of nutrient starvation, hypoxia, increased
ATP utilization, or heat shock. This mammalian enzyme is composed of a cat
alytic a:subunit and noncatalytic beta and gamma subunits and is a member o
f a larger protein kinase family that includes the SNF1 kinase of Saccharom
yces cerevisiae. In the present study, we have identified by truncation and
site-directed mutagenesis several functional domains of the alpha 1 cataly
tic subunit, which modulate its activity, subunit association, and protein
turnover. C-terminal truncation of the 548-amino acid (aa) wild-type alpha
1 protein to aa 312 or 392 abolishes the binding of the beta/gamma subunits
and dramatically increases protein expression. The full-length wild-type a
lpha 1 subunit is only minimally active in the absence of co-expressed beta
/gamma, and alpha 1(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 activ
ator, AMP. The increased levels of protein expression on transient transfec
tion of either truncated a: subunit cDNA are because of a decrease in enzym
e turnover by pulse-chase analysis. Taken together, these data indicate tha
t the alpha 1 subunit of AMP-activated protein kinase contains several feat
ures 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-activ
ated protein kinase.