Colin M. House

Mammalian AMP-activated Protein Kinase Subfamily

The mammalian 5′-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (α1) is distinct from the previously cloned kinase subunit (α2). The α1 (548 residues) and α2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase α1 mRNA distribution and the α1 immunoreactivity rather than α2, with substantial amounts in kidney, liver, lung, heart, and brain. Both α1 and α2 isoforms are stimulated by AMP and contain noncatalytic β and γ subunits. The liver α1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the α2 immunoreactivity parallels the α2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the β and γ subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.

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.

IL6-STAT3-HIF Signaling and Therapeutic Response to the Angiogenesis Inhibitor Sunitinib in Ovarian Clear Cell Cancer

Purpose: Ovarian clear cell adenocarcinoma (OCCA) is an uncommon histotype that is generally refractory to platinum-based chemotherapy. We analyze here the most comprehensive gene expression and copy number data sets, to date, to identify potential therapeutic targets of OCCA. Experimental Design: Gene expression and DNA copy number were carried out using primary human OCCA tumor samples, and findings were confirmed by immunohistochemistry on tissue microarrays. Circulating interleukin (IL) 6 levels were measured in serum from patients with OCCA or high-grade serous cancers and related to progression-free and overall survival. Two patients were treated with sunitinib, and their therapeutic responses were measured clinically and by positron emission tomography. Results: We find specific overexpression of the IL6-STAT3-HIF (interleukin 6-signal transducer and activator of transcription 3-hypoxia induced factor) pathway in OCCA tumors compared with high-grade serous cancers. Expression of PTHLH and high levels of circulating IL6 in OCCA patients may explain the frequent occurrence of hypercalcemia of malignancy and thromboembolic events in OCCA. We describe amplification of several receptor tyrosine kinases, most notably MET, suggesting other potential therapeutic targets. We report sustained clinical and functional imaging responses in two OCCA patients with chemotherapy-resistant disease who were treated with sunitinib, thus showing significant parallels with renal clear cell cancer. Conclusions: Our findings highlight important therapeutic targets in OCCA, suggest that more extensive clinical trials with sunitinib in OCCA are warranted, and provide significant impetus to the growing realization that OCCA is molecularly and clinically distinct to other forms of ovarian cancer. Clin Cancer Res; 17(8); 2538–48. ©2011 AACR.

Isoform-specific purification and substrate specificity of the 5'-AMP-activated protein kinase.

The 5′-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either α1 or α2 catalytic subunits together with β and γ noncatalytic subunits in a trimeric complex. The α1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK α1 with a Km of 3.8 μM and a Vmax of 4.8 μmol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 μM and a Vmax of 8.1 μmol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK α1 isoform has a Kcat ∼250-fold higher than the AMPK α2 isoform isolated from rat liver. The AMPK α1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Φ(X, β)XXS/TXXXΦ (Φ, hydrophobic; β, basic). In good AMPK α1 peptide substrates, a hydrophobic residue at the P−5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.

Posttranslational Modifications of the 5′-AMP-activated Protein Kinase β1 Subunit

The AMP-activated protein kinase (AMPK) consists of catalytic α and noncatalytic β and γ subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK α1 and α2 catalytic subunits are associated with β1 and γ1 noncatalytic subunits. We find that the isolated γ1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated β1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable β1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated β1subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylatedin vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that α1 Thr172 is a major, although not exclusive, site of both basal and stimulated α1phosphorylation by an upstream AMPK kinase.

Deregulation of MYCN, LIN28B and LET7 in a molecular subtype of aggressive high-grade serous ovarian cancers

Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.

A binding motif for Siah ubiquitin ligase

The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including β-catenin. Despite these diverse targets, the means by which SINA/Siah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95–117). The unusually high-affinity binding obtained with a 23-residue peptide (KDapp = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINA/Siah proteins.

Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling.

Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 Å resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded β-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-α receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-α-mediated NF-κB activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.

Substrate and pseudosubstrate interactions with protein kinases: determinants of specificity

Protein crystallography has revealed that protein kinases have extended protein-substrate-binding grooves associated with their active sites. Some protein kinases are autoinhibited by a mechanism in which part of their structure, termed a pseudosubstrate, occupies the active site. Substrates and pseudosubstrates occupy overlapping regions within the extended substrate-binding groove, making multiple specific electrostatic and non-polar contacts. With masterly economy, Nature has exploited the active site in many protein kinases to both recognize substrates with great specificity and autoregulate by remaining inactive until the appropriate activation signal is received.

Phosphorylation at the Cyclin-dependent Kinases Site (Thr85) of Parathyroid Hormone-related Protein Negatively Regulates Its Nuclear Localization

Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61–94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10, 34, 35) for PTHrP.