Muscle contractions, AICAR, and insulin cause phosphorylation of an AMPK-related kinase.
We hypothesized that AMP-activated protein kinase-related kinase 5 (ARK5)/novel kinase family 1 (NUAK1), an AMP-activated protein kinase (AMPK)-related kinase that has been found to be stimulated by protein kinase B (Akt), would be expressed in rat skeletal muscle and activated by electrically elicited contractions, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), or insulin. We verified expression of ARK5 in muscle through RT-PCR and Western blot. Cross-reactivity of ARK5 immunoprecipitates with antibodies against phospho-AMPK was increased by approximately 30% by muscle contractions and approximately 60% by incubation of muscle with AICAR. AMPK was not detected in the ARK5 immunoprecipitates. Despite the apparent increase in phosphorylation of ARK5 at a site essential to its activation, neither contractions nor AICAR increased ARK5 activity. For muscles from animals injected with saline or insulin, we probed nonimmunoprecipitated samples in sequence for phosphotyrosine (P-Tyr), ARK5, and phosphorylated substrates of Akt (P-AS) and found that the ARK5 band could be precisely superimposed on phosphoprotein bands from the P-Tyr and P-AS blots. In the band corresponding to ARK5, insulin increased P-Tyr content by approximately 45% and cross-reactivity with the antibody against P-AS by approximately threefold. We also detected ARK5 in phosphotyrosine immunoprecipitates. Our data suggest that increased phosphorylation of ARK5 by muscle contractions or exposure to AICAR is insufficient to activate ARK5 in skeletal muscle, suggesting that some other modification (e.g., phosphorylation on tyrosine or by Akt) may be necessary to its activity in muscle.
Fisher
Jonathan S
JS
Dept. of Biology, Saint Louis University, 3507 Laclede Avenue, St. Louis, MO 63103, USA. fisherjs@slu.edu
Ju
Jeong-Sun
JS
Oppelt
Peter J
PJ
Smith
Jill L
JL
Suzuki
Atsushi
A
Esumi
Hiroyasu
H
D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012265:Ribonucleotides / Q000494:pharmacology*
D007004:Hypoglycemic Agents; D007328:Insulin; D012333:RNA, Messenger; D012097:Repressor Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D011494:Protein Kinases; C470845:NUAK1 protein, human; C506345:Nuak1 protein, rat; C031143:AICA ribonucleotide