Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2003-01-31
2004-12-21
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S254110, C435S320100, C435S325000, C435S410000, C435S455000, C536S023100, C536S023200, C536S023500, C530S350000
Reexamination Certificate
active
06833255
ABSTRACT:
The present invention relates to a protein kinase enzyme, specifically to p70 S6 kinase (p70
S6K
) isolated from
Drosophila melanogaster.
In the signal transduction pathway mediating the multiple phosphorylation of 40S ribosomal protein S6 in response to mitogens and oncogenes, the most proximal signalling components to S6 are p70
S6K
and p85
S6K
(Ferrari, S. & Thomas, G. (1994) CRC Crit. Rev. Biochem. Mol. Biol. 29, 385-413; Jefferies, H. B. J. & Thomas, G. (1995) in Translational Control, eds. Hershey, J. W. B., Mathews, M. B. & Sonenberg, N., Cold Spring Harbor Press, Cold Spring Harbor). Both of these kinase isoforms are generated from a common mRNA transcript through the use of alternative initiation translation start sites, residing within 23 codons of one another. The additional 23 amino acid extension at the amino terminus of p85
S6K
contains a nuclear localisation sequence, which constitutively targets this isoform to the nucleus, whereas p70
S6K
appears to be exclusively cytoplasmic. Consistent with the localisation of S6 in both compartments of the cell, the available data suggest that both kinase isoforms are regulated in a closely coordinated fashion. Little is known regarding the functional role of p85
S6K
and phosphorylated S6 in the nucleus, however recent studies employing the immunosuppressant rapamycin have led to the hypothesis that p70
S6K
, through increased S6 phosphorylation, is involved in the selective translational upregulation of a family of mRNAs characterised by a oligopyrimidine tract at their 5′ transcriptional start sites. Although these mRNAs, termed 5′ TOP, comprise a small family, they can represent up to 20-30% of the cells mRNA transcripts, and in most cases code for essential gene products of the translational apparatus, such as ribosomal proteins.
The link between mitogen-induced p70
S6K
activation and the translational upregulation of 5′ TOP mRNAs is based on the selective effects of the rapamycin-FKBP12 gain-of-function inhibitory complex on both responses. Activation of p70
S6K
is associated with the phosphorylation of the enzyme at multiple residues, which exhibit two distinct phosphorylation motifs. The sites initially identified are flanked by a proline in the +1 position (Ferrari, S., et al. (1992) Proc. Nat i. Acad. Sci. USA 89, 7282-7285) and, with the exception of S411, are rapamycin resistant. The second set of sites is flanked in the +1 and −1 positions by large aromatic residues, and exhibit rapamycin sensitivity (Pearson, R. B., et al. (1995) EMBO J. 14, 5279-5287). Of these latter sites the principal target of rapamycin-induced p70
S6K
inactivation and dephosphorylation is T389 in the linker region, that couples the catalytic and autoinhibitory domains. Conversion of this site to an acidic residue confers rapamycin resistance and constitutive activity on the kinase. Truncation mutants of p70
S6K
have revealed, that in the absence of the amino-terminus, rapamycin can no longer block mitogen-induced T389 phosphorylation or kinase activation, demonstrating that inhibitory effects of rapamycin are not exerted through blocking the activation of an upstream kinase. However the mechanism by which rapamycin affects p70
S6K
activation through the amino-terminus remains unresolved. The failure to identify the immediate upstream p70
S6K
kinases as well as the mechanism by which rapamycin regulates p70
S6K
activation is largely explained by the multiple events required to bring about this response and the failure to reconstitute these events in vitro. This failure prompted the application of a number of complementary indirect approaches, including the use of inhibitors (Chung, J., et al. (1994) Nature 370, 71-75), dominant negative signalling molecules (Ming, X. F., et al. (1994) Nature 371, 426-429), and growth factor receptor mutants in the search for upstream components that regulate this response. However, as yet, this strategy has not yielded any additional insight into the identity of the upstream p70
S6K
kinases nor the mechanism for the selective effects of rapamycin on the kinase.
A powerful system which could complement the phosphorylation site studies described above, as well as resolve conflicting issues concerning the involvement of specific signalling molecules, is the use of developmental genetics. The use of genetic systems has proven extremely important in establishing the components that make up specific signal transduction pathways. For example, the application of a genetic approach to the study of the
Drosophila
sevenless mutant played a direct role in elucidating the signalling components of the MAP kinase pathway. In the case of p70
S6K
, this system offers the additional attraction that in
Drosophila
, unlike yeast and slime moulds, the majority of ribosomal proteins contain a 5′TOP and their expression is selectively regulated at the translational level, in a manner similar to that found in mammals. Thus, the identification of a
Drosophila
p70
S6K
homologue could prove very important in identifying immediate upstream kinases and downstream targets as well as the mechanism by which rapamycin influences these responses.
SUMMARY OF THE INVENTION
The invention accordingly provides
Drosophila melanogaster
p70
S6K
, as well as nucleic acids encoding this kinase. The sequence of
Drosophila
p70
S6K
and the gene encoding it are represented in SEQ ID No. 2 and 1 respectively. The invention moreover provides mutated forms of
Drosophila
p70
S6K
, including constitutively active and dominant negative forms thereof, which are useful in the study of p70
S6K
activity. Furthermore, the invention provides expression systems which produce
Drosophila
p70
S6K
in
Drosophila
and other organisms, and in particular systems in which expression of
Drosophila
p70
S6K
has been modulated so as to facilitate the study of its activity.
REFERENCES:
patent: 0239400 (1987-03-01), None
patent: WO 9007861 (1990-07-01), None
Banerjee, et al., Proc. Natl. Acad. Sci. USA, “Molecular Structure of a Major Insulin/Mitogen-activated 70-kDa S6 Protein Kinase”, vol. 87, pp. 8550-8554 (1990).
Baumgartner, et al., Genes & Dev., “Structure of Two Genes at the Gooseberry Locus Related to the Paired Gene and Their Spatial Expression During Drosophila Embryogenesis”, vol. 1, pp. 1247-1267 (1987).
Blenis, et al, Proc. Natl. Acad. Sci., “Signal Transduction Via the Map Kinases: Proceed at Your Own RSK”, vol. 90, pp. 5889-5892 (1993).
Chen, C.A. & Okayama H., BioTechniques, vol. 6 (7), “Calcium Phosphate-Mediated Gene Transfer: A Highly Efficient Transfection System for Stably Transforming Cells with Plasmid DNA,” pp. 632-638 (1998).
Chung J. et al., Nature, vol. 370, “PDGF-and insulin-dependent pp70.sup.S6K activation mediated by phosphatidylinositol-3-OH kinase,” pp. 71-75 (1994).
Dennis P.B. et al., Mol. & Cell. Biol., vol. 16 (11), “The Principal Rapamycin-Sensitive p70.sup.S6K Phosphorylation Sites, T-229 and T-389, Are Differentially Regulated by Rapamycin-Insensitive Kinase Kinases,”.
Downward J., Nature, vol. 376, “A target for PI(3) kinase,” pp. 553-554 (1995).
Ferrari, S. et al., Proc. Natl. Acad. Sci. USA, vol. 89, “Activation of p70.sup.S6K is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs,” pp. 7282-7287 (1992).
Ferrari S. et al., J. Biol. Chem., vol. 268 (22), “The Immunosuppressant Rapamycin Induces Inactivation of p70.sup.S6K through Dephosphorylation of a Novel set of Sites,” pp. 16091-16094 (1993).
Ferrari and Thomas, Crit. Rev. in Biochem. and Mol. Biol., vol. 29 (6), “S6 Phosphorylation and the p70.sup.S6K ,” pp. 385-413 (1994).
Flotow H. & Thomas G., J. Biol. Chem., vol. 267 (5), “Substrate Recognation Determinants of the Mitogen-activated 70K S6 Kinase from Rat Liver,” pp. 3074-3078 (1992).
Grove J.R. et al., Molecular and Cell Biology, vol. 11 (11), “Cloning and Expression of Two Human p70 S6 Kinase Polypeptides Differing Only at Their Amino Termini,” pp. 5541-5550 (1991).
Hahn C. et al., Proc. Natl. Acad. Sci. USA, vol. 89,
Kozma Sara
Stewart Mary
Thomas George
Kaushal Sumesh
Leffers Gerry
Novartis AG
Prince John T.
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