AMP-activated protein kinase - not just an energy sensor

Orthologues of AMP-activated protein kinase (AMPK) occur in essentially all eukaryotes as heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits. The canonical role of AMPK is as an energy sensor, monitoring levels of the nucleotides AMP, ADP, and ATP that bind competitively to the γ subunit. Once activated, AMPK acts to restore energy homeostasis by switching on alternate ATP-generating catabolic pathways while switching off ATP-consuming anabolic pathways. However, its ancestral role in unicellular eukaryotes may have been in sensing of glucose rather than energy. In this article, we discuss a few interesting recent developments in the AMPK field. Firstly, we review recent findings on the canonical pathway by which AMPK is regulated by adenine nucleotides. Secondly, AMPK is now known to be activated in mammalian cells by glucose starvation by a mechanism that occurs in the absence of changes in adenine nucleotides, involving the formation of complexes with Axin and LKB1 on the surface of the lysosome. Thirdly, in addition to containing the nucleotide-binding sites on the γ subunits, AMPK heterotrimers contain a site for binding of allosteric activators termed the allosteric drug and metabolite (ADaM) site. A large number of synthetic activators, some of which show promise as hypoglycaemic agents in pre-clinical studies, have now been shown to bind there. Fourthly, some kinase inhibitors paradoxically activate AMPK, including one (SU6656) that binds in the catalytic site. Finally, although downstream targets originally identified for AMPK were mainly concerned with metabolism, recently identified targets have roles in such diverse areas as mitochondrial fission, integrity of epithelial cell layers, and angiogenesis

Overexpression of FK-506-binding protein 12.0 modulates excitation-contraction coupling in adult rabbit ventricular cardiomyocytes

The effect of the 12-kDa isoform of FK-506-binding protein (FKBP)12.0 on cardiac excitation contraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6 +/- 0.4 fold, n = 5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S-transferase pull-down experiments indicated FKBP12.0-RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. Voltage-clamp experiments indicated no effects of FKBP12.0 overexpression on L-type Ca2+ current (I-Ca,I-L) or Ca2+ efflux rates via the Na+/Ca2+ exchanger. Ca2+ transient amplitude was also not significantly different. However, sarcoplasmic reticulum Ca2+ load was approximate to 25% higher in myocytes in the Ad-FKBP12.0 group. The reduced ability of ICa, L to initiate sarcoplasmic reticulum Ca2+ release was observed over a range of values of sarcoplasmic reticulum Ca2+ content, indicating that overexpression of FKBP12.0 reduces the sensitivity of RyR2 to Ca2+. Ca2+ spark morphology was measured in beta-escin-permeabilized cardiomyocytes. Ca2+ spark amplitude and duration were significantly increased, whereas frequency was decreased in cells overexpressing FKBP12.0. These changes were accompanied by an increased sarcoplasmic reticulum Ca2+ content. In summary, the effects of FKBP12.0 overexpression on intact and permeabilized cells were similar to those of tetracaine, a drug known to reduce RyR2 Ca2+ sensitivity and distinctly different from the effects of overexpression of the FKBP12.6 isomer. In conclusion, FKBP12.0-RyR2 interaction can regulate the gain of excitation-contraction coupling

Increased sarcoplasmic reticulum calcium leak but unaltered Contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes

The predominant cardiac Ca2+/calmodulin-dependent protein kinase (CaMK) is CaMKII delta. Here we acutely overexpress CaMKII delta(C) using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII delta(C) transgenic mice. CaMKII delta(C) protein expression and activation state ( autophosphorylation) were increased 5- to 6- fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKII delta(C) versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced (tau(0.5Hz)/tau(3Hz)=2.14 +/- 0.10 versus 1.87 +/- 0.10), and peak Ca2+ current (I-Ca) was increased by 31% (-7.1 +/- 0.5 versus -5.4 +/- 0.5 pA/pF, P < 0.05). Ca2+ transient amplitude was not significantly reduced (-27%, P = 0.22), despite dramatically reduced sarcoplasmic reticulum (SR) Ca2+ content (41%; P < 0.05). Thus fractional SR Ca2+ release was increased by 60% (P < 0.05). Diastolic SR Ca2+ leak assessed by Ca2+ spark frequency (normalized to SR Ca2+ load) was increased by 88% in CaMKII delta(C) versus LacZ myocytes (P < 0.05; in an multiplicity-of-infection-dependent manner), an effect blocked by CaMKII inhibitors KN-93 and autocamtide-2-related inhibitory peptide. This enhanced SR Ca2+ leak may explain reduced SR Ca2+ content, despite measured levels of SR Ca2+-ATPase and Na+/Ca2+ exchange expression and function being unaltered. Ryanodine receptor (RyR) phosphorylation in CaMKII delta(C) myocytes was increased at both Ser2809 and Ser2815, but FKBP12.6 coimmunoprecipitation with RyR was unaltered. This shows for the first time that acute CaMKII delta(C) overexpression alters RyR function, leading to enhanced SR Ca2+ leak and reduced SR Ca2+ content but without reducing twitch contraction and Ca2+ transients. We conclude that this is attributable to concomitant enhancement of fractional SR Ca2+ release in CaMKII delta(C) myocytes (ie, CaMKII-dependent enhancement of RyR Ca2+ sensitivity during diastole and systole) and increased ICa.NHLBI NIH HHS [HL-28143, HL-30077, HL-46345, HL-64724