TY - JOUR
T1 - Regulation of brain-type creatine kinase by AMP-activated protein kinase: Interaction, phosphorylation and ER localization
AU - Rios, Sacnicte Ramirez
AU - Lamarche, Frederic
AU - Cottet-Rousselle, Cecile
AU - Klaus, Anna
AU - Tuerk, Roland
AU - Thali, Ramon
AU - Auchli, Yolanda
AU - Brunisholz, Rene
AU - Neumann, Dietbert
AU - Barret, Luc
AU - Tokarska-Schlattner, Malgorzata
AU - Schlattner, Uwe
PY - 2014/8
Y1 - 2014/8
N2 - AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca?-pumping.
AB - AMP-activated protein kinase (AMPK) and cytosolic brain-type creatine kinase (BCK) cooperate under energy stress to compensate for loss of adenosine triphosphate (ATP) by either stimulating ATP-generating and inhibiting ATP-consuming pathways, or by direct ATP regeneration from phosphocreatine, respectively. Here we report on AMPK-dependent phosphorylation of BCK from different species identified by in vitro screening for AMPK substrates in mouse brain. Mass spectrometry, protein sequencing, and site-directed mutagenesis identified Ser6 as a relevant residue with one site phosphorylated per BCK dimer. Yeast two-hybrid analysis revealed interaction of active AMPK specifically with non-phosphorylated BCK. Pharmacological activation of AMPK mimicking energy stress led to BCK phosphorylation in astrocytes and fibroblasts, as evidenced with a highly specific phospho-Ser6 antibody. BCK phosphorylation at Ser6 did not affect its enzymatic activity, but led to the appearance of the phosphorylated enzyme at the endoplasmic reticulum (ER), close to the ER calcium pump, a location known for muscle-type cytosolic creatine kinase (CK) to support Ca?-pumping.
KW - Protein kinase
KW - Creatine kinase
KW - Endoplasmic reticulum
KW - Energy homeostasis
KW - Protein phospholylation
KW - Subcellular localization
U2 - 10.1016/j.bbabio.2014.03.020
DO - 10.1016/j.bbabio.2014.03.020
M3 - Article
C2 - 24727412
SN - 0005-2728
VL - 1837
SP - 1271
EP - 1283
JO - Biochimica et Biophysica Acta-bioenergetics
JF - Biochimica et Biophysica Acta-bioenergetics
IS - 8
ER -