Ian G. Jennings

AMPK β Subunit Targets Metabolic Stress Sensing to Glycogen

Abstract AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients [1], as well as hormones, including adiponectin and leptin [2, 3]. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK [4]. Mammalian AMPK is an αβγ heterotrimer with multiple isoforms of each subunit comprising α1, α2, β1, β2, γ1, γ2, and γ3, which have varying tissue and subcellular expression [5, 6]. Mutations in the AMPK γ subunit cause glycogen storage disease in humans [7], but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK β subunit contains a functional glycogen binding domain (β-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished β-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK β1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.

Structural basis of autoregulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an α-β sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.

Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.

Turn up the HEAT.

The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats. The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.

Phosphorylation regulates copper-responsive trafficking of the Menkes copper transporting P-type ATPase

The Menkes copper-translocating P-type ATPase (ATP7A) is a critical copper transport protein functioning in systemic copper absorption and supply of copper to cuproenzymes in the secretory pathway. Mutations in ATP7A can lead to the usually lethal Menkes disease. ATP7A function is regulated by copper-responsive trafficking between the trans-Golgi Network and the plasma membrane. We have previously reported basal and copper-responsive kinase phosphorylation of ATP7A but the specific phosphorylation sites had not been identified. As copper stimulates both trafficking and phosphorylation of ATP7A we aimed to identify all the specific phosphosites and to determine whether trafficking and phosphorylation are linked. We identified twenty in vivo phosphorylation sites in the human ATP7A and eight in hamster, all clustered within the N- and C-terminal cytosolic domains. Eight sites were copper-responsive and hence candidates for regulating copper-responsive trafficking or catalytic activity. Mutagenesis of the copper-responsive phosphorylation site Serine-1469 resulted in mislocalization of ATP7A in the presence of added copper in both polarized (Madin Darby canine kidney) and non-polarized (Chinese Hamster Ovary) cells, strongly suggesting that phosphorylation of specific serine residues is required for copper-responsive ATP7A trafficking to the plasma membrane. A constitutively phosphorylated site, Serine-1432, when mutated to alanine also resulted in mislocalization in the presence of added copper in polarized Madin Darby kidney cells. These studies demonstrate that phosphorylation of specific serine residues in ATP7A regulates its sub-cellular localization and hence function and will facilitate identification of the kinases and signaling pathways involved in regulating this pivotal copper transporter.

Essential role of the N-terminal autoregulatory sequence in the regulation of phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH4). The crystal structure of PAH revealed that the N‐terminal sequence of the enzyme (residues 19–29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N‐terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH4. Our data support the model where the N‐terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.

Structure/function analysis of the domains required for the multimerisation of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) exists as an equilibrium of dimers and tetramers. However, there is little information concerning the inter- or intra-molecular interactions required for enzyme quaternary structure. It is predicted that the formation of a PAH tetramer will require at least two points of contact per enzyme subunit. Sequence analysis has suggested the existence of a C-terminal domain with characteristics of a leucine zipper or a variant of this called a coiled-coil. By deletion of 24 amino acids from the C-terminus or conversion of leucine 448 to an alanine residue, we have shown that this putative leucine zipper/coiled-coil domain is involved in the assembly of an active enzyme tetramer from dimers. The removal of this C-terminal domain of PAH reduces enzyme activity but does not abolish it. Furthermore, we report that an alanine 447 to aspartate mutation associated with phenylketonuria may affect subunit assembly which suggests the formation of enzyme tetramers is physiologically relevant. Our analysis of subunit interactions in vivo, show that in the absence of the C-terminal coiled-coil domain, dimers can form and this is only possible when the N-terminal domain is present. This provides the first evidence that N-terminal domain is required for multimerisation. We propose that the N-terminal regulatory domain in conjunction with the C-terminal coiled-coil domain, mediates the formation of fully active enzyme tetramers.

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP‐dependent protein kinase at Ser16 in the N‐terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N‐terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N‐terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N‐terminal region, but no sharp resonances are present in a truncation mutant lacking the N‐terminal 29 residues. The N‐terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2–20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N‐terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Structure and Regulation of Phenylalanine Hydroxylase, and Implications for Related Enzymes

Phenylalanine hydroxylase (PAH) is a metabolic enzyme that converts Phe to Tyr using molecular oxygen, enzyme-bound iron, and a 6R-tetrahydrobiopterin (BH4) cofactor (1, 2, 3). PAH is a member of the aromatic amino acid hydroxylase family, together with tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH). TH and TPH are involved in the biosynthesis of the neurotransmitters, L-DOPA and serotonin, respectively. The aromatic amino acid hydroxylases share a similar enzyme mechanism and have a common three-domain structure consisting of an N-terminal regulatory domain, a catalytic domain and a C-terminal tetramerization domain; the highest sequence and structural similarity is found in the catalytic domain. Over 300 different mutations in the PAH gene have been found to be associated with the disease phenylketonuria (PKU) (4), although only a small proportion of mutant proteins have been functionally characterised (5).