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Biomedical subjects

T F Franke

Publications and source records attributed to T F Franke.

33 records · Page 2Linked to original sources
PubMed1998

Role of Akt and c-Jun N-terminal kinase 2 in apoptosis induced by interleukin-4 deprivation.

We have shown previously that interleukin-4 (IL-4) protects TS1alphabeta cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1alphabeta cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1alphabeta survival is independent of Bcl-2, Bcl-x, or Bax.

Animals↗
PubMed1997

Interleukin 3-dependent survival by the Akt protein kinase.

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.

Animals↗
PubMed1997

Regulation of neuronal survival by the serine-threonine protein kinase Akt.

A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70(S6K)). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70(S6K) mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.

Androstadienes↗
PubMed1997

Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate.

The regulation of the serine-threonine kinase Akt by lipid products of phosphoinositide 3-kinase (PI 3-kinase) was investigated. Akt activity was found to correlate with the amount of phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) in vivo, and synthetic PtdIns-3,4-P2 activated Akt both in vitro and in vivo. Binding of PtdIns-3,4-P2 occurred within the Akt pleckstrin homology (PH) domain and facilitated dimerization of Akt. Akt mutated in the PH domain was not activated by PI 3-kinase in vivo or by PtdIns-3, 4-P2 in vitro, and it was impaired in binding to PtdIns-3,4-P2. Examination of the binding to other phosphoinositides revealed that they bound to the Akt PH domain with much lower affinity than did PtdIns-3,4-P2 and failed to increase Akt activity. Thus, Akt is apparently regulated by the direct interaction of PtdIns-3,4-P2 with the Akt PH domain.

3T3 Cells↗
PubMed1995

The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase.

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.

Androstadienes↗
PubMed1995

AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

Amino Acid Sequence↗
PubMed1994

The SH2-like Akt homology (AH) domain of c-akt is present in multiple copies in the genome of vertebrate and invertebrate eucaryotes. Cloning and characterization of the Drosophila melanogaster c-akt homolog Dakt1.

The Akt proto-oncogene encodes a serine-threonine protein kinase whose carboxyterminal catalytic domain is closely related to the catalytic domains of all the known members of the protein kinase C (PKC) family. Akt, however, differs from PKC in its N-terminal region which contains a domain related distantly to the SH2 domain of cytoplasmic tyrosine kinases and other signalling proteins, which we have named Akt homology (AH) domain. Low stringency hybridization of a c-akt AH probe to a panel of genomic DNAs from vertebrate and invertebrate eucaryotes detected multiple DNA bands (perhaps multiple genes) in all tested species. Drosophila DNA contains at least three hybridizing DNA bands. One of them was cloned, and found by sequence analysis, to define an Akt related gene (Dakt1). Comparison between the coding regions of c-akt and Dakt1 revealed 64.6% identity at the nucleotide level and 76.5% similarity at the amino acid level. The highest degree of homology was detected in the AH domain (68.3% similarity at the amino acid level) and the catalytic domain (83.3% similarity). Additional sequence comparisons revealed that the amino acid similarity between the catalytic domains of Dkt1 and the three known members of the Drosophila protein kinase C (PKC) family, Dpkc1, Dpkc2 and Dpkc3, is 68%, 63.6% and 67.1%, respectively. Dakt1 was mapped to Drosophila chromosome 3R 89BC. Its expression is subject to developmental regulation with the highest levels detected within the fourth hour of embryonic development. These results confirm that the AH domain of Akt defines new protein families in both vertebrate and invertebrate eucaryotes. The high degree of homology between the catalytic domains of Dkt1 and the three known members of the Drosophila PKC family suggests an evolutionarily conserved functional relationship between the members of the two families.

Amino Acid Sequence↗
PubMed1993

Structure, expression and chromosomal mapping of c-akt: relationship to v-akt and its implications.

Sequence analysis of a nearly full-length murine c-akt cDNA clone and comparison with v-akt revealed the following: (a) The entire coding region of c-akt is identical to that of v-akt with the exception of five G to A transitions that do not alter the reading frame. The 3' untranslated regions of v-akt and c-akt are also identical with the exception of three single-base differences. (b) The recombination event that gave rise to v-akt occurred between the virus at nucleotide 785 from the Gag ATG codon and the 5' untranslated region of c-akt to 60 bp 5' from the c-akt ATG codon. (c) Three nucleotides absent from both Gag and c-akt were inserted at the junction between the two genes. The outcome of these events was to place, in frame, a 63-bp fragment between Gag and Akt. The resulting v-akt oncogene is predicted to encode a tripartite Gag (p12, p15, delta p30)-X-c-akt protein product. The c-akt protein contains, starting from its amino terminus, a src homology 2-like (SH2-like) domain, a domain rich in glutamic acid residues, part of which is predicted to form an amphipathic helix, and a kinase domain encoding a serine-threonine kinase with high degree of homology to members of the protein kinase C (PKC) family. The mouse c-akt is 90% homologous to human AKT1/RAC at the nucleic acid level and 98% homologous at the amino acid level. c-akt in the mouse is composed of 13 exons. The first exon contains a 5' untranslated GC-rich region. Since the recombination that gave rise to v-akt occurred with the 5' untranslated region, we hypothesize that the transduction of c-akt was preceded by provirus insertion upstream from or within the 5' untranslated region and in the same transcriptional orientation as the gene. c-akt was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 12 and rat chromosome 6 in close proximity to the Igh locus.

Amino Acid Sequence↗
PubMed1993

The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential.

The acute retrovirus AKT8, isolated from an AKR mouse T-cell lymphoma, transforms mink lung cells in culture and is oncogenic when inoculated into newborn mice. The oncogene carried by this virus, v-akt, arose by recombination between Gag and the 5' untranslated region of the cellular gene c-akt. v-akt encodes a 105 kilodalton (kd) Gag-Akt fusion protein which is phosphorylated on serine and threonine residues. c-akt encodes a 55 kd serine-threonine protein-kinase, which is related to members of the protein kinase C (PKC) family and contains an SH2-like domain. The SH2-like and catalytic domains of Akt were expressed in E. coli as fusions to the carboxy-terminus of the Maltose binding protein (MBP). Antibodies against these proteins were raised in rabbits and they were used to determine the potential myristylation and subcellular localization of the v-akt and c-akt protein products. Immunoprecipitation of v-akt and c-akt from lysates of [35S]methionine and [3H]myristic acid labeled AKT8 transformed mink lung cells revealed that only v-akt was myristylated. Fractionation of Dounce-homogenized cellular extracts from uninfected and v-akt-transformed mink lung and PA317 cells and from uninfected PC12 cells by differential centrifugation showed that while the c-akt protein was localized primarily in the cytosol (90%), the v-akt protein was dispersed among the cellular compartments with approximately 40% on the plasma membranes, approximately 30% in the nucleus and approximately 30% in the cytosol. To determine whether the differences in post-translational modification and subcellular distribution between c-akt and v-akt translated into oncogenicity differences between the two proteins, we used retrovirus based constructs to express them both in the nontumorigenic rat T cell lymphoma line 5675. Intraperitoneal (IP) inoculation of the parental and c-akt expressing 5675 cells in nude Balb/c mice revealed that neither was oncogenic. In sharp contrast to these results, v-akt expressing 5675 cells inoculated in nude Balb/c mice were found to be highly oncogenic.

Amino Acid Sequence↗
PubMed1992

AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas.

We isolated cDNA clones containing the entire coding region of the putative oncogene AKT2. Sequence analysis and in vitro translation demonstrated that AKT2 encodes a 56-kDa protein with homology to serine/threonine kinases; moreover, this protein contains a Src homology 2-like domain. AKT2 was shown to be amplified and overexpressed in 2 of 8 ovarian carcinoma cell lines and 2 of 15 primary ovarian tumors. AKT2 was mapped to chromosome region 19q13.1-q13.2 by fluorescence in situ hybridization. In the two ovarian carcinoma cell lines exhibiting amplification of AKT2, the amplified sequences were localized within homogeneously staining regions. We conclude that AKT2 belongs to a distinct subfamily of protein-serine/threonine kinases containing Src homology 2-like domains and that alterations of AKT2 may contribute to the pathogenesis of ovarian carcinomas.

Amino Acid Sequence↗