AMP-activated protein kinase (AMPK) plays an important role in maintaining energy homeostasis in mammals. AMPK is heterotrimer of an α catalytic subunit, and two regulatory subunits, β and γ. In mammals, each subunit has different isoforms (α1/α2, β1/β2, and γ1/γ2/γ3) encoded by separate genes leading to the potential expression of 12 AMPK complexes. Here we show that AMPK containing the long forms of γ2 (γ2a, encoding a protein of 569 amino acids, and γ2c, 525 amino acids) bind to 14-3-3. In contrast to AMPK containing the short form of γ2 (γ2b, 328 amino acids), bacterial expression of AMPK containing the long forms of g2 requires co-expression with 14-3-3 and prior phosphorylation of Thr172 within the α subunit. AMPKγ2-14-3-3 complexes have reduced activity compared to AMPKγ1 or AMPKγ2b but retain allosteric activation by AMP and the AMPK activator, 991. We found that two predicted 14-3-3 binding sites within γ2a (T97 and S122) were phosphorylated in the bacterially expressed AMPK complex. Furthermore, we show that a peptide spanning these two phosphorylated sites binds to 14-3-3 in vitro and determined the crystal structure of this 14-3-3-peptide co-complex. These results indicate that 14-3-3 binds to the N-terminal region of γ2a/c, reducing the activity of AMPK relative to AMPKγ1 and AMPKγ2b. Our findings reveal a new mode of regulation of AMPK containing the long forms of γ2. Whilst the biological significance of 14-3-3 binding to AMPKγ2a/c complexes remains to be determined, our studies provide the starting point to begin to address this issue.
CARLING et al. (Fri,) studied this question.