Biochem. J. (2001) 354
(275–283) (Printed in Great Britain)
Post-translational modifications of the b-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization
Scott M. WARDEN*, Christine RICHARDSON*, John O'DONNELL Jr*, David STAPLETON†, Bruce E. KEMP† and Lee A. WITTERS*1
*Endocrine-Metabolism Division, Departments of Medicine and Biochemistry, Dartmouth Medical School, Remsen 322, N. College St, Hanover, NH 03755, U.S.A., and †St Vincent's Institute of Medical Research, 41Victoria Parade, Fitzroy, Victoria 3065, Australia
The AMP-activated protein kinase (AMPK) is a ubiquitous mammalian protein kinase important in the adaptation of cells to metabolic stress. The enzyme is a heterotrimer, consisting of a catalytic a subunit and regulatory b and g subunits, each of which is a member of a larger isoform family. The enzyme is allosterically regulated by AMP and by phosphorylation of the a subunit. The b subunit is post-translationally modified by myristoylation and multi-site phosphorylation. In the present study, we have examined the impact of post-translational modification of the b-1 subunit on enzyme activity, heterotrimer assembly and subcellular localization, using site-directed mutagenesis and expression of subunits in mammalian cells. Removal of the myristoylation site (G2A mutant) results in a 4-fold activation of the enzyme and relocalization of the b subunit from a particulate extranuclear distribution to a more homogenous cell distribution. Mutation of the serine-108 phosphorylation site to alanine is associated with enzyme inhibition, but no change in cell localization. In contrast, the phosphorylation site mutations, SS24,25AA and S182A, while having no effects on enzyme activity, are associated with nuclear redistribution of the subunit. Taken together, these results indicate that both myristoylation and phosphorylation of the b subunit of AMPK modulate enzyme activity and subunit cellular localization, increasing the complexity of AMPK regulation.
Key words: AMP-activated protein kinase, enzyme subunit structure, myristoylation and phosphorylation.
Abbreviations used: AMPK, AMP-activated protein kinase; AMPKK, AMPK kinase; CMV, cytomegalovirus; DMEM, Dulbecco's minimal essential medium; GFP, green fluorescent protein; GST, glutathione S transferase; YFP, yellow fluorescent protein.
1To whom correspondence should be addressed (e-mail lee.a.witters@dartmouth.edu).
Received 13 September 2000/15 December 2000; accepted 3 January 2001
The Biochemical Society, London ©
2001
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