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Activation of the Snf1 kinase
requires at
least two events, phosphorylation of the activation loop on threonine
210 and an Snf4-dependent process that is not completely defined. Snf4
directly interacts with a region of the regulatory domain of Snf1 that
may otherwise act as an autoinhibitory domain. In order to gain insight
into the regulation of Snf1 kinase by Snf4, deletions in the regulatory
domain of the catalytic subunit were engineered and tested for their
effect on Snf1 function in the absence of Snf4. Deletion of residues 381
to 488 from the Snf1 protein resulted in a kinase that was activated by
glucose limitation even in the absence of the Snf4 protein. A larger
deletion (amino acids 381 to 608) encompassing virtually the entire
regulatory domain resulted in complete inactivation of the Snf1 kinase
even in the presence of Snf4. A genetic screen for amino acid
substitutions that conferred an Snf4-independent phenotype identified
four point mutations in the Snf1 catalytic domain. One very conservative
mutation, leucine 183 to isoleucine, conferred nearly wild-type levels
of Snf1 kinase function in the absence of the Snf4 protein. Purified
Snf1 kinase was inactive when isolated from snf4Δ cells,
whereas the
Snf1-L183I kinase exhibited significant activity in the absence of Snf4.
Our data support the idea that Snf1 kinase activity is constrained in
cis by an autoinhibitory domain and that the Snf4-mediated activation of
Snf1 can be bypassed by subtle conformational changes in the catalytic
domain of the Snf1 kinase. |
