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Regulation of brain-type creatine kinase by AMP-activated protein kinase: interaction, phosphorylation and ER localization


Ramírez Ríos, Sacnicte; Lamarche, Frédéric; Cottet-Rousselle, Cécile; Klaus, Anna; Tuerk, Roland; Thali, Ramon; Auchli, Yolanda; Brunisholz, René; Neumann, Dietbert; Barret, Luc; Tokarska-Schlattner, Malgorzata; Schlattner, Uwe (2014). Regulation of brain-type creatine kinase by AMP-activated protein kinase: interaction, phosphorylation and ER localization. BBA - Biochimica et Biophysica Acta, 1837(8):1271-1283.

Abstract

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 Ca²⁺-pumping.

Abstract

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 Ca²⁺-pumping.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:August 2014
Deposited On:29 Jan 2015 11:45
Last Modified:05 Apr 2016 18:59
Publisher:Elsevier
ISSN:0006-3002
Publisher DOI:https://doi.org/10.1016/j.bbabio.2014.03.020
PubMed ID:24727412

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