Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

BACKGROUND: Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. METHODS: Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. RESULTS: The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. CONCLUSIONS: For the first time this data associates altered CDK5 substrate phosphorylation with oncogenesis in some but not all tumour types, implicating altered CDK5 activity in aspects of pathogenesis. These data identify a novel oncogenic mechanism where CDK5 activation induces CRMP2A phosphorylation in the nuclei of tumour cells.

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice.

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.

The calcium binding protein ALG-2 binds and stabilizes Scotin, a p53-inducible gene product localized at the endoplasmic reticulum membrane.

ALG-2 (apoptosis linked gene 2 product) is a calcium binding protein for which no clear cellular function has been established. In this study we identified Scotin as a novel ALG-2 target protein containing 6 PXY and 4 PYP repeats, earlier identified in the ALG-2 binding regions of AIP1/ALIX and TSG101, respectively. An in vitro synthesized C-terminal fragment of Scotin bound specifically to immobilized recombinant ALG-2 and tagged ALG-2 and Scotin were shown by immunoprecipitation to interact in MCF7 and U2OS cell lines. Furthermore ALG-2 bound to endogenous Scotin in extracts from mouse NIH3T3 cells. Overexpression of ALG-2 led to accumulation of Scotin in MCF7 and H1299 cells. In vitro and in vivo binding of ALG-2 to Scotin was demonstrated to be strictly calcium dependent indicating a role of this interaction in calcium signaling pathways.

Characterization of a protein kinase B inhibitor in vitro and in insulin-treated liver cells.

OBJECTIVE: Abnormal expression of the hepatic gluconeogenic genes (glucose-6-phosphatase [G6Pase] and PEPCK) contributes to hyperglycemia. These genes are repressed by insulin, but this process is defective in diabetic subjects. Protein kinase B (PKB) is implicated in this action of insulin. An inhibitor of PKB, Akt inhibitor (Akti)-1/2, was recently reported; however, the specificity and efficacy against insulin-induced PKB was not reported. Our aim was to characterize the specificity and efficacy of Akti-1/2 in cells exposed to insulin and then establish whether inhibition of PKB is sufficient to prevent regulation of hepatic gene expression by insulin. RESEARCH DESIGN AND METHODS: Akti-1/2 was assayed against 70 kinases in vitro and its ability to block PKB activation in cells exposed to insulin fully characterized. RESULTS: Akti-1/2 exhibits high selectivity toward PKBalpha and PKBbeta. Complete inhibition of PKB activity is achieved in liver cells incubated with 1-10 mumol/l Akti-1/2, and this blocks insulin regulation of PEPCK and G6Pase expression. Our data demonstrate that only 5-10% of maximal insulin-induced PKB is required to fully repress PEPCK and G6Pase expression. Finally, we demonstrate reduced insulin sensitivity of these gene promoters in cells exposed to submaximal concentrations of Akti-1/2; however, full repression of the genes can still be achieved by high concentrations of insulin. CONCLUSIONS: This work establishes the requirement for PKB activity in the insulin regulation of PEPCK, G6Pase, and a third insulin-regulated gene, IGF-binding protein-1 (IGFBP1); suggests a high degree of functional reserve; and identifies Akti-1/2 as a useful tool to delineate PKB function in the liver.

p14 Arf promotes small ubiquitin-like modifier conjugation of Werners helicase.

Here we demonstrate a novel p53-independent interaction between the nucleolar tumor suppressors, p14 Arf and Werners helicase (WRN). Binding of p14 Arf to WRN is multivalent and resembles the binding of p14 Arf to Mdm2. Residues 2-14 and 82-101 of p14 Arf and residues in the central region and C terminus of WRN have particular importance for binding. p14 Arf promotes small ubiquitin-like modifier (SUMO) modification of WRN in a synergistic manner with the SUMO-conjugating enzyme, UBCH9. p14 Arf causes redistribution of WRN within the nucleus, and this effect is reversed by expression of a SUMO-specific protease, thus implicating the SUMO conjugation pathway in WRN re-localization. We establish that the ability to promote SUMO conjugation is a general property of the p14 Arf tumor suppressor.

Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo.

p53 levels are regulated by ubiquitination and 26 S proteasome-mediated degradation. p53 is a substrate for the E3 ligase Mdm2, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination in intact cells have not been defined previously. To investigate the E2 specificity of Mdm2 we carried out an in vitro screen using a panel of ubiquitin E2s. Of the E2s tested only UbcH5A, -B, and -C and E2-25K support Mdm2-mediated ubiquitination of p53. The same E2s also support Mdm2 auto-ubiquitination. Small interfering RNA-mediated knockdown of UbcH5B/C causes accumulation of Mdm2 and p53 in unstressed cells. We show that suppression of UbcH5B/C inhibits p53 ubiquitination and degradation. Despite up-regulating the level of nuclear p53, UbcH5B/C knockdown does not on its own result in an increase in p53 transcriptional activity or sensitize p53 to activation by the therapeutic drugs doxorubicin and actinomycin D. We provide evidence that Mdm2 is responsible, at least in part, for repression of the transcriptional activity of the accumulated p53. In MCF7 cells levels of UbcH5B/C are reduced by doxorubicin and actinomycin D. This observation and the sensitivity of p53 expression to levels of UbcH5B/C raise the possibility that E2 regulation could be involved in signaling pathways that control the stability of p53. Our data indicate that UbcH5B/C are physiological E2s for Mdm2, which make a significant contribution to the maintenance of low levels of p53 and Mdm2 in unstressed cells and that inhibition of p53 ubiquitination and degradation by targeting UbcH5B/C is not sufficient to up-regulate p53 transcriptional activity.

Exploiting the p53 pathway for cancer diagnosis and therapy.

Cancer research has reached an exciting phase of its evolution, as we now have an increasingly detailed molecular description of the genomic abnormalities and the biochemical pathways that drive the malignant progression of cancers. Investigation of the p53 tumour suppressor pathway, in particular, has become a key focus of current cancer research. This review describes how an increasing knowledge of the biochemistry of the p53 pathway is allowing us to develop novel strategies that rationally manipulate the function of this pathway in tumour cells for therapeutic benefit. Work in this field represents an outstanding illustration of how basic scientific research is being translated into clinical applications and the many challenges this involves.

Two novel phosphorylation sites on FKHR that are critical for its nuclear exclusion.

FKHR is phosphorylated by protein kinase B (PKB) at Thr24, Ser256 and Ser319 in response to growth factors, stimulating the nuclear exit and inactivation of this transcription factor. Here we identify two further residues, Ser322 and Ser325, that become phosphorylated in insulin-like growth factor-1 (IGF-1)-stimulated cells and which are mediated by the phosphatidylinositol 3-kinase-dependent PKB-catalysed phosphorylation of Ser319. Phosphorylation of Ser319 forms a consensus sequence for phosphorylation by CK1, allowing it to phosphorylate Ser322, which in turn primes the CK1-catalysed phosphorylation of Ser325. IGF-1 stimulates the phosphorylation of Thr24, Ser256, Ser319, Ser322 and Ser325 in embryonic stem (ES) cells, but not in PDK1-/- ES cells, providing genetic evidence that PDK1 (the upstream activator of PKB) is required for the phosphorylation of FKHR in mammalian cells. In contrast, the phosphorylation of Ser329 is unaffected by IGF-1 and the phosphorylation of this site is not decreased in PDK1-/- ES cells. The cluster of phosphorylation sites at Ser319, Ser322, Ser325 and Ser329 appears to accelerate nuclear export by controlling the interaction of FKHR with the Ran-containing protein complex that mediates this process.