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

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.

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites

“…It consists of at least four distinct spots with same molecular weight but different isoelectric points. The pI shift is typical for phosphorylation events, and indeed BCK autophosphorylation (Hemmer et al 1995) and phosphorylation by protein kinase C (PKC; Chida et al 1990) or AMP-activated protein kinase (AMPK; Ponticos et al 1998;Ramirez Rios et al 2014) have been reported in literature, but was unrelated to mitochondria (Ramirez Rios et al 2014). MS-MS analysis confirmed that all four spots in 2D-PAGE correspond to BCK, but was unable so far to identify a post-translational modification (results not shown).…”

“…Perfused hypoxic transgenic liver with BCK was capable of maintaining global Fig. 4 BCK localization at the ER calcium pump is regulated by phosphorylation via AMPK (Ramirez Rios et al 2014). a BCK from different species in vitro phosphorylated by AMPK.…”

“…4b), which is exposed at the surface of the BCK dimer (Fig. 4f) and conserved as a Ser or Thr residue across vertebrate species (Ramirez Rios et al 2014). This phosphorylation is not affecting BCK enzyme activity as reported in earlier literature for MCK (Ponticos et al 1998), but localizes BCK to perinuclear regions identified as ER in fibroblasts and astrocytes, in close proximity to the ER Ca 2+ ATPase pump (SERCA; Fig.…”

“…Interaction with metallothioneins protects BCK against oxidative and nitrosative molecular damage (Chen et al 2012b), and binding to hippocalcin may translocate BCK to membranes in a Ca 2+ -dependent manner (Kobayashi et al 2012). A transient interaction of BCK with AMP-activated protein kinase (AMPK) that regulates BCK localization depending on the cellular energy state (Ramirez Rios et al 2014) is further discussed in "Regulation of BCK localization by phosphorylation".…”

“…4a; Ramirez Rios et al 2014). AMPK is a central cellular signaling hub that, among others, senses a deteriorated cellular energy state, characterized by a rise in [ADP] and [AMP] (Hardie et al 2011;Oakhill et al 2012;Chen et al 2012a), and then initiates compensatory measures to improve ATP supply (in general via an increase in catabolic pathways) Fig.…”