Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure-function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.

Previous studies showed that insulin antagonizes AMP-activated protein kinase activation by ischemia and that protein kinase B might be implicated. Here we investigated whether the direct phosphorylation of AMP-activated protein kinase by protein kinase B might participate in this effect. Protein kinase B phosphorylated recombinant bacterially expressed AMP-activated protein kinase heterotrimers at Ser 485 of the ␣1-subunits. In perfused rat hearts, phosphorylation of the ␣1/␣2 AMP-activated protein kinase subunits on Ser 485 /Ser 491 was increased by insulin and insulin pretreatment decreased the phosphorylation of the ␣-subunits at Thr 172 in a subsequent ischemic episode. It is proposed that the effect of insulin to antagonize AMP-activated protein kinase activation involves a hierarchical mechanism whereby Ser 485 /Ser 491 phosphorylation by protein kinase B reduces subsequent phosphorylation of Thr 172 by LKB1 and the resulting activation of AMP-activated protein kinase.Stimulation of the insulin and AMP-activated protein kinase (AMPK) 4 signaling pathways in heart leads to an increase in glycolysis via recruitment of GLUT4 transporters to the plasma membrane and activation of 6-phosphofructo-2-kinase (PFK-2) (1, 2). The signaling pathway for insulin requires phosphatidylinositol 3-kinase (PI3K) and, for PFK-2 activation, protein kinase B (PKB), and/or a wortmanninsensitive and insulin-stimulated protein kinase that phosphorylates heart PFK-2 on Ser 466 (3). PFK-2 activation in ischemia is explained by the activation of AMPK, which also phosphorylates heart PFK-2 at Ser 466 (2).AMPK is a heterotrimer consisting of a catalytic ␣-subunit together with two regulatory subunits, ␤ and ␥. Each subunit exists as multiple isoforms (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, ␥3) giving 12 different possible combinations of holoenzyme with different tissue distribution and subcellular localization (4 -6). In heart, the ␣2 isoform of the catalytic subunit is twice as abundant as ␣1 (7). The activating upstream AMPK kinase (AMPKK) phosphorylating Thr 172 in the AMPK catalytic ␣-subunits was initially partially purified from rat liver and reported to contain a M r 58,000 catalytic subunit in a M r 195,000 complex (8). Site-directed mutagenesis showed that phosphorylation at Thr 172 accounts for most of the activation of AMPK by AMPKK; however, new phosphorylation sites were identified as Thr 258 and Ser 485/491 in the catalytic ␣1/␣2-subunits, respectively (9). Site-directed mutagenesis experiments indicated that phosphorylation at these new sites was not essential for AMPK activation or activity, whereas Thr 172 was required (9). Recently, one AMPK-activating AMPKK phosphorylating Thr 172 was identified as the Peutz-Jeghers syndrome protein LKB1 (10, 11). LKB1 can phosphorylate the activation loop Thr residue of several members of the AMPK family (12).In perfused heart, ischemia antagonizes insulin signaling via a drop in pH, which inhibits the tyrosine kinase activity of the insulin receptor (13). By contrast, pretreatment with insulin ...