Carboxyl-terminal Modulator Protein Research Articles

Regulation of OPA1-mediated mitochondrial fusion by leucine zipper/EF-hand-containing transmembrane protein-1 plays a role in apoptosis

Carboxyl-terminal modulator protein (CTMP) is a tumor suppressor-like binding partner of Protein kinase B (PKB/Akt) that negative regulates this kinase. In the course of our recent work, we identified that CTMP is consistently associated with leucine zipper/EF-hand-containing transmembrane-1 (LETM1). Here, we report that adenovirus-LETM1 increased the sensitivity of HeLa cells to apoptosis, induced by either staurosporine or actinomycin D. As shown previously, LETM1 localized to the inner mitochondrial membrane. Electron-microscopy analysis of adenovirus-LETM1 transduced cells revealed that mitochondrial cristae were swollen in these cells, a phenotype similar to that observed in optic atrophy type-1 (OPA1)-ablated cells. OPA1 cleavage was increased in LETM1-overexpressing cells, and this phenotype was reversed by overexpression of OPA1 variant-7, a cleavage resistant form of OPA1. Taken together, these data suggest that LETM1 is a novel binding partner for CTMP that may play an important role in mitochondrial fragmentation via OPA1-cleavage.

High dietary inorganic phosphate increases lung tumorigenesis and alters Akt signaling.

Phosphate (Pi) is an essential nutrient to living organisms. Recent surveys indicate that the intake of Pi has increased steadily. Our previous studies have indicated that elevated Pi activates the Akt signaling pathway. An increased knowledge of the response of lung cancer tissue to high dietary Pi may provide an important link between diet and lung tumorigenesis. The current study was performed to elucidate the potential effects of high dietary Pi on lung cancer development. Experiments were performed on 5-week-old male K-ras(LA1) lung cancer model mice and 6-week-old male urethane-induced lung cancer model mice. Mice were fed a diet containing 0.5% Pi (normal Pi) and 1.0% Pi (high Pi) for 4 weeks. At the end of the experiment, all mice were killed. Lung cancer development was evaluated by diverse methods. A diet high in Pi increased lung tumor progression and growth compared with normal diet. High dietary Pi increased the sodium-dependent inorganic phosphate transporter-2b protein levels in the lungs. High dietary consumption of Pi stimulated pulmonary Akt activity while suppressing the protein levels of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 as well as Akt binding partner carboxyl-terminal modulator protein, resulting in facilitated cap-dependent protein translation. In addition, high dietary Pi significantly stimulated cell proliferation in the lungs of K-ras(LA1) mice. Our results showed that high dietary Pi promoted tumorigenesis and altered Akt signaling, thus suggesting that careful regulation of dietary Pi may be critical for lung cancer prevention as well as treatment.

470. Aerosol Delivery of Carboxyl-Terminal Modulator Protein (CTMP) Regulated Apoptosis and Angiogenesis in the Lungs of K-Ras LA1 Mice

470. Aerosol Delivery of Carboxyl-Terminal Modulator Protein (CTMP) Regulated Apoptosis and Angiogenesis in the Lungs of K-Ras LA1 Mice

Lentivirus-mediated carboxyl-terminal modulator protein gene transfection via aerosol in lungs of K-ras null mice

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Aerosol gene delivery may provide the alternative for safe and effective treatment for lung cancer. Therefore, current study was performed to elucidate the potential effects of C-terminal modulator protein (CTMP) via aerosol on lung tumorigenesis. Lentiviral vector-CTMP was delivered into K-ras null lung cancer mice through the nose-only inhalation system for 30 min. After 48 h, the potential effects of CTMP on Akt1-related signals and cell cycle regulation in the lungs were evaluated by western blot, immunohistochemistry and zymography. Lentivirus-based CTMP delivery inhibited the Akt1 activity through selective suppression of Akt1 phosphorylation at Ser473. Aerosol delivery of CTMP inhibited proteins important for Akt1 signals, cell cycle and tumor metastasis in lungs of K-ras null mice. Together, our results suggest that lentivirus-mediated aerosol delivery of CTMP may be compatible with noninvasive in vivo gene therapy. Our results emphasize the importance of noninvasive-targeted delivery of CTMP for lung cancer therapy in the future. While the studies are conducted in mice, it is envisioned that noninvasive targeting the specific genes responsible for cancer progression is an attractive strategy for effective anticancer therapeutics.

Activation of Akt independent of PTEN and CTMP tumor-suppressor gene mutations in epilepsy-associated Taylor-type focal cortical dysplasias

Focal cortical dysplasias (FCD) with Taylor-type balloon cells (FCD(IIb)) are frequently observed in biopsy specimens of patients with pharmacoresistant focal epilepsies. The molecular pathogenesis of FCD(IIb), which lack familial inheritance, is only poorly understood. Due to their highly differentiated, malformative nature and glioneuronal phenotype, FCD(IIb) share neuropathological characteristics with lesions observed in familial disorders such as cortical tubers present in patients with autosomal dominant tuberous sclerosis complex (TSC), related to mutations in the TSC1 or TSC2 genes, and dysplastic gangliocytomas of the cerebellum found in Cowden disease. Current data have indicated distinct allelic variants of TSC1 to accumulate in FCD(IIb). TSC1 represents a tumor suppressor operating in the phosphatidylinositol 3-kinase (PI3K)/insulin pathway. The tumor-suppressor gene PTEN is mutated in Cowden disease. Like PTEN, also carboxyl-terminal modulator protein (CTMP) modulates PI3K-pathway signaling, both via inhibition of Akt/PKB, a kinase inactivating the TSC1/TSC2 complex. Here, we have analyzed alterations of Akt, PTEN and CTMP relevant for insulin signaling upstream of TSC1/TSC2 in FCD(IIb). Immunohistochemistry with antibodies against phosphorylated Akt (phospho-Akt; Ser 473) in FCD(IIb) (n=23) showed strong phospho-Akt expression in dysplastic FCD(IIb) components. We have further studied sequence alterations of PTEN (n=34 FCD(IIb)) and CTMP (n=20 FCD(IIb)) by laser microdissection/single-strand conformation polymorphism analysis. We observed a somatic mutation in an FCD(IIb), i.e., amino-acid exchange at nucleotide position 834 (PTEN cDNA, GenBank AH007803.1) in exon 8 with replacement of phenylalanine by leucine (F278L). We also found several silent polymorphisms of PTEN in exon 2 and exon 8 as well as silent and coding polymorphisms but no mutations in CTMP. No loss of heterozygosity in FCD(IIb) (n=6) at 10q23 was observed. To our knowledge, we here report on the first somatic mutation of a tumor-suppressor gene, i.e., PTEN, in FCD(IIb). However, our study also demonstrates that mutational alterations of PTEN and CTMP do not play major pathogenetic roles for activation of Akt in FCD(IIb). Future studies need to determine the origin of insulin pathway activation upstream of TSC1/TSC2 in FCD(IIb).

Genetic alteration and expression of the phosphoinositol‐3‐kinase/Akt pathway genes PIK3CA and PIKE in human glioblastomas

Glioblastomas frequently carry genetic alterations resulting in an aberrant activation of the phosphoinositol-3-kinase (Pi3k)/protein kinase B (Akt) signalling pathway, including most notably phosphatase and tensin homolog (PTEN) mutation, epidermal growth factor receptor (EGFR) amplification and rearrangement, as well as carboxyl-terminal modulator protein (CTMP) hypermethylation [Knobbe et al., (2004) Hypermethylation and transcriptional downregulation of the carboxyl-terminal modulator protein gene in glioblastomas. J Natl Cancer Institute, 96, 483-486]. Here, we investigated two further Pi3k/Akt pathway genes, namely PIK3CA (3q26.3) and phosphatidylinositol-3-kinase enhancer (PIKE) (CENTG1, 12q14), for genetic alteration and aberrant expression in a series of 97 primary glioblastomas. Single strand conformation polymorphism (SSCP) analysis of PIK3CA revealed somatic mutations in five tumours (5%). Twelve glioblastomas (12%) showed amplification of PIKE with invariable co-amplification of the adjacent CDK4 gene. All tumours with PIKE amplification as well as the vast majority of glioblastomas without amplification demonstrated increased expression of PIKE-A but not PIKE-S/L transcripts as compared with non-neoplastic brain tissue. Taken together, our data support an important role of PIK3CA and PIKE gene aberrations in the molecular pathogenesis of primary glioblastomas.

Hypermethylation and transcriptional downregulation of the carboxyl-terminal modulator protein gene in glioblastomas.

The carboxyl-terminal modulator protein (CTMP) has been identified as a negative regulator of protein kinase B/Akt. Aberrant Akt signaling is frequently observed in glioblastomas, the most common and most malignant glial brain tumors. Because loss of CTMP function and/or expression may remove the inhibitory effects on Akt and promote tumorigenesis, we studied 93 primary glioblastomas and nine glioblastoma cell lines for CTMP deletion, mutation, promoter hypermethylation, and mRNA expression. None of the tumors or cell lines had CTMP-homozygous deletions or coding sequence mutations. However, CTMP mRNA expression was lower by at least 50% relative to non-neoplastic brain tissue in 37 (40%) glioblastomas and six (67%) glioma cell lines. Reduced CTMP mRNA levels were closely associated with hypermethylation of the CTMP promoter. Furthermore, treatment of CTMP-hypermethylated A172 glioma cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in partial demethylation of the CTMP promoter and increased CTMP mRNA expression. Thus, epigenetic downregulation of CTMP transcription is a common aberration in glioblastomas.

Keeping the Bad Guy Down: New potential tumor-suppressor gene

In the good guy-bad guy world of cancer, shifty proteins often run afoul of cellular law, prompting molecular sheriffs to action. One cellular citizen--the protein Akt--normally lives a quiet life, performing its civic duty in the insulin-like growth factor-1 (IGF-1) and insulin mammalian signaling pathways. These pathways manage cell growth in mammals, and related ones regulate aging in organisms that include worms, flies, and possibly mice (see "Growing Old Together" ). To do its job, Akt slips on a phosphate group. If the protein dons this black hat for too long, however, it triggers a molecular stampede in the IGF-1 signaling pathway, which promotes a half-dozen cancers. Mutations in Akt or its handlers can turn Akt to a permanent life of crime. A corrupt form of the protein, found in many cancers, never removes its phosphate; this version transforms innocuous cell lines into deadly tumor inducers in mice. In healthy cells, a white-hatted cellular cowboy--a tumor-suppressor gene known as PTEN --keeps Akt in check. Research has now uncovered a second marshal in the mammalian struggle against unrestrained Akt that keeps the phosphate hat away from the potential scoundrel, preventing it from suiting up in its gunslinger garb. To seek regulators of Akt, Hemmings and colleagues used a technique to fish out molecules that bind to a bait protein--in this case, a piece of Akt--from a pool of human proteins. Using a variety of biochemical techniques, they showed that one protein--which they named CTMP--prevents Akt from acquiring the phosphate group that leads to its hyperactivity. They then produced CTMP in a cell line made cancerous by the delinquent Akt. CTMP quashed the ability of these cells to grow uncontrolled in culture dishes and reduced the speed at which they induce tumors in mice. These results suggest a rationale for new cancer therapies: Perhaps a drug that mimics CTMP could wrangle in a lawless Akt. The researchers also found that the amount of CTMP in cells mirrored that of PTEN and propose that the two might be regulated in parallel. If so, CTMP could be partly responsible for Akt lawlessness that is now blamed on a slacking PTEN. CTMP provides a new focus for labs that are trying to discover how IGF-1-like signaling pathways control life-span. Perhaps this molecular desperado plays a role not just in cancer but also in longevity: It might help decide whether to send cells--or organisms--riding off into the sunset. --Mary Beckman S.-M. Maira, I. Galetic, D. P. Brazil, S. Kaech, E. Ingley, M. Thelen, B. A. Hemmings, Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science 294 , 374-380 (2001). [Abstract] [Full Text]

Regulating Protein Kinase B

Protein kinase B (PKB) has proven to be a key regulatory element in growth factor signaling, cell survival, and transformation to a cancerous phenotype. Maira et al. screened for proteins that physically interacted with PKB and identified a human protein, which they call carboxyl-terminal modulator protein (CTMP), that binds to PKB at the plasma membrane and inhibits activation of the kinase. Inhibition of CTMP expression enhanced activation of PKB, whereas cells expressing a viral homolog of PKB became transformed and proliferated abnormally and could form tumors in vivo. However, clones of such cells stably expressing CTMP grew more slowly and either failed to form tumors in vivo or formed tumors that grew more slowly. Thus, CTMP appears to be an important regulator of PKB, an enzyme with critical roles from insulin signaling to protection of cells from apoptosis.S.-M. Maira, I. Galetic, D. P. Brazil, S. Kaech, E. Ingley, M. Thelen, B. A. Hemmings, Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science 294 374-380 (2001). [Abstract] [Full Text]

Regulating Protein Kinase B

Protein kinase B (PKB) has proven to be a key regulatory element in growth factor signaling, cell survival, and transformation to a cancerous phenotype. Maira et al. screened for proteins that physically interacted with PKB and identified a human protein, which they call carboxyl-terminal modulator protein (CTMP), that binds to PKB at the plasma membrane and inhibits activation of the kinase. Inhibition of CTMP expression enhanced activation of PKB, whereas cells expressing a viral homolog of PKB became transformed and proliferated abnormally and could form tumors in vivo. However, clones of such cells stably expressing CTMP grew more slowly and either failed to form tumors in vivo or formed tumors that grew more slowly. Thus, CTMP appears to be an important regulator of PKB, an enzyme with critical roles from insulin signaling to protection of cells from apoptosis. S.-M. Maira, I. Galetic, D. P. Brazil, S. Kaech, E. Ingley, M. Thelen, B. A. Hemmings, Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science 294 374-380 (2001). [Abstract] [Full Text]

Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane.

The PKB (protein kinase B, also called Akt) family of protein kinases plays a key role in insulin signaling, cellular survival, and transformation. PKB is activated by phosphorylation on residues threonine 308, by the protein kinase PDK1, and Serine 473, by a putative serine 473 kinase. Several protein binding partners for PKB have been identified. Here, we describe a protein partner for PKBalpha termed CTMP, or carboxyl-terminal modulator protein, that binds specifically to the carboxyl-terminal regulatory domain of PKBalpha at the plasma membrane. Binding of CTMP reduces the activity of PKBalpha by inhibiting phosphorylation on serine 473 and threonine 308. Moreover, CTMP expression reverts the phenotype of v-Akt-transformed cells examined under a number of criteria including cell morphology, growth rate, and in vivo tumorigenesis. These findings identify CTMP as a negative regulatory component of the pathway controlling PKB activity.