Colin M. House

The regulatory domain of protein kinase C contains an amino acid sequence between residues 19 and 36 that resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants. The corresponding synthetic peptide (Arg19-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val-His -Glu-Val-Lys-Asn36) acts as a potent substrate antagonist with an inhibitory constant of 147 +/- 9 nM. It is a specific inhibitor of protein kinase C and inhibits both autophosphorylation and protein substrate phosphorylation. Substitution of Ala25 with serine transforms the pseudosubstrate into a potent substrate. These results demonstrate that the conserved region of the regulatory domain (residues 19 to 36) of protein kinase C has the secondary structural features of a pseudosubstrate and may be responsible for maintaining the enzyme in the inactive form in the absence of allosteric activators such as phospholipids.

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. 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.

The AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. It may also regulate cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. We have purified the AMP-activated protein kinase 14,000-fold from porcine liver. The 63-kDa catalytic subunit co-purifies with two proteins of 40 and 38 kDa that may function as subunits. Partial amino acid sequence of the 63-kDa subunit revealed a striking homology with the catalytic domain of the yeast protein kinase transcriptional regulator Snf1 and its plant homologs. The Snf1 (72 kDa) and Snf4 (36 kDa) complex was also purified and found to phosphorylate the AMP-activated protein kinase peptide substrate, HMRSAMSGLHLVKRR-amide, but was not activated by AMP. Both Snf1/4 and the AMP-activated protein kinase phosphorylate and inactivate yeast acetyl-CoA carboxylase in vitro. These results indicate that during evolution the catalytic domain sequences of the Snf1 protein kinase subfamily have been exploited in the control of mammalian lipid metabolism and raise the possibilities that the AMP-activated protein kinase may have other substrates involved in regulating gene expression pathways, as well as Snf1 homologs participating in the control of lipid metabolism in many eukaryotic organisms.

Abstract TP53 , a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11 , a component of the cystine/glutamate antiporter, system x C − , through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System x C − inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system x C − antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11–glutathione axis.

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.