The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.
The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.
Departments of Medicine and Biochemistry, Dartmouth Medical School and the Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.
Rebecca L
RL
Hurley
Kristin A
KA
Anderson
Jeanne M
JM
Franzone
Bruce E
BE
Kemp
Anthony R
AR
Means
Lee A
LA
Witters
D000091162:AMP-Activated Protein Kinase Kinases; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003847:Deoxyglucose / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D015759:Ionomycin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008353:Mannitol / Q000737:chemistry; D051379:Mice; D053644:Naphthalimides; D010766:Phosphorylation; D020033:Protein Isoforms; D011494:Protein Kinases / Q000737:chemistry / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry
D001562:Benzimidazoles; D002147:Calmodulin; D016895:Culture Media, Serum-Free; D007546:Isoquinolines; D053644:Naphthalimides; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D012265:Ribonucleotides; C458525:STO 609; D013912:Threonine; D000620:Aminoimidazole Carboxamide; D008353:Mannitol; D015759:Ionomycin; D003847:Deoxyglucose; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases; C031143:AICA ribonucleotide; D002118:Calcium