Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1998-11-23
2001-01-30
Longton, Enrique D. (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S183000, C435S069100, C530S350000
Reexamination Certificate
active
06180380
ABSTRACT:
The present invention relates to a new serine threonine kinase, the gene coding therefor and the detection of changes in this gene and its products in human tumours.
The activation of intracellular biochemical networks as a response to external stimuli leads to coordinated control of growth and of differentiation in eukaryotes. Protein kinases are known as constituents of many signal transduction pathways. In this connection, these kinases phosphorylate their normal physiological substrates and are regulated in their enzymatic activity by interaction with other kinases and phosphatases. The identification of a large number of protein kinases in many eukaryotic cells of mammals, yeast and Drosophila makes it probable that findamental cellular differentiation and growth processes are controlled by identical mechanisms in a wide spectrum of organisms.
In eukaryotes, all protein kinases known until now phosphorylate the hydroxyamino acids serine, threonine or tyrosine. Phosphorylation by these kinases plays a prominent role in the control of mitosis and cellular differentiation. Receptors for numerous polypeptide growth factors are transmernbrane tyrosine kinases which, for their parts, phosphorylate serine/threonine kinases such as protein kinase C, MP kinase and p74raf The central component of the cell cycle machinery is the serine/threonine kinase p34cdc2, which has originally been isolated as a product of the mitosis gene cdc2 (cell division cycle) from
Schizosaccharomyces pombe
and CDC28 from
Saccharomyces cerevisiae
. The activity of p34cdc2 is regulated by interaction with cyclins. In the G1 phase, the start of the cell cycle, p34cdc2 is not associated with cyclins and has no kinase activity. If cells are supplied with sufficient nutrients, G1 cyclin accumulates which, by association, initiates the kinase activity of p34cdc2. By this means “start” is exceeded and the change in the cell cycle (DNA replication, formation of the MTOC, microtubule organizing centre) is initiated. In this connection, the synthesis of cyclin B also begins, which then binds to p34cdc2. This complex is inactive, since the binding of cyclin B induces the phosphorylation of p34cdc2 on tyrosine 15, whereby the kinase activity is inhibited. The inactive complex, which is also called preMPF (maturation promoting factor), is also phosphorylated on threonine 160. This phosphorylation is necessary for the MPF activity, but not sufficient in order to abolish the inhibitory (effects of tyrosine phosphorylation. The subsequent dephosphorylation of p34cdc2 during the late G2 phase of the cell cycle activates MPF and leads to the induction of mitosis (M phase). The post-translational reactions, which underlie a complex physiological control, play an essential part in the temporal regulation of the MPF activity. The protein tyrosine kinase weel was originally isolated from
Schizosaccharomyces pombe
and inhibits, via the mechanism described, entry into mitosis, while the product of the cdc25 gene promotes the start of mitosis. Active MPF activates tyrosine phosphatase and inhibits protein tyrosine kinase, which modify p34cdc2, whereby MPF is completely activated explosively, such that cells are driven into mitosis very rapidly and irreversibly.
The most recent investigations of the cell cycle show that essential regulators of the cell cycle are involved in carcinogenesis. This is not surprising because constant proliferation of cells is an outstanding feature of tumours. Changes to cyclin A, which binds both to p34cdc2 and to p34cdc2-related protein kinase, can cause the transfornation of cells. The cyclin A gene is the integration site for a fragment of the hepatitis B virus genome in a human hepatocarcinoma. Moreover, cyclin A is associated with the transforming protein E1A in adenovirus-transformed cells. Cyclin A is possibly a target protein of E1A, since it is associated in the S phase with the transcription factor E2F in a complex which has lower transcription activity than free E2F. A further constituent of this complex is p34cdc2. The connection with gene expression is produced in this way. E1A can destroy this complex, whereby E2F is released. Thus certain genes can be regulated which are important for the transformed phenotype.
Moreover, there are further relationships between oncoproteins, tumour suppressor gene products and cyclin-p34cdc2 complexes. The mos oncoprotein is likewise a serine/threonine kinase. The c-mos protein from Xenopus is a component of the cytostatic factor, which is necessary for the stabilization of the activated cyclin B-p34cdc2 in growth-inhibited Xenopus eggs. The role of the mos protein, however, is still unclear, as it does not appear to phosphorylate the complex in vivo. On the one hand, c-mos stabilizes the activated cyclin B-P34cdc2 complex, whereby the mitosis activity of the cells is inhibited, and on the other hand it promotes the cell cycle. Previous investigations confirm the assumption that the differing behaviour of the cell cycle machinery is controlled by the amount of mos protein.
Various oncoproteins such as the src and abl protein tyrosine kinases are likewise phosphorylated by the serinelthreonine kinase p34cdc2 in the context of mitosis. In the src family, mitotic phosphorylation is accompanied by increased kinase activity. The products of the tumour suppressor genes RB and p53 likewise form complexes with cyclin-p34cdc2 and are phosphorylated in this process. In the case of RB, the phosphorylation appears to be necessary in order to inactivate the RB function so that the cells of G1 can progress into the S phase. As a consequence of the aberrant expression of the cyclin-p34cdc2 complex, it follows that such tumour suppressor gene products are preserved in their phosphorylated inactive state, which results in unchecked cell division.
The connection between the function of serine/threonine kinases of the cell cycle and the transformation shows clearly that slight disturbances in the mitosis processes play a critical part in carcinogenesis. As carcinogenesis appears to be a multi-stage process, mutations in cell cycle regulators cooperate with mutations which activate protooncogenes or inactivate tumour suppressor genes.
On account of the enormous clinical importance of serine threonine kinases, there was thus a need to make available a new serine threonine kinase and the gene coding therefor.
The invention thus relates to a new serine threonine kinase called PLK, which is characterized in that it
(a) comprises the amino acid sequence shown in SEQ ID No: 2 or
(b) variants of the sequence from (a).
Preferably, the PLK protein according to the invention is a protein obtainable from man, i.e. it is the protein shown in SEQ ID No. 1 and No. 2 or a naturally occurring human variant thereof.
The invention also relates to a new protein which comprises parts of the amino acid sequence shown in SEQ ID No: 1 and 2. The invention preferably relates to a PLK protein which contains the amino acid sequence shown in SEQ ID No: 1 and 2; however, it can also contain variants of this sequence. The term “variants” within the meaning of the present invention is understood as meaning sequences which differ as a result of substitution, deletion and/or insertion of individual amino acids or short amino acid sections from the amino acid sequence shown in SEQ ID No: 1 and 2.
The term “variants” includes both naturally occurring allelic variations of the PLK protein, as well as proteins produced by recombinant DNA technology (in particular by in vitro mutagenesis with the aid of chemically synthesized oligonucleotides), which (correspond with respect to their biological and/or immunological activity to the protein shown in SEQ ID No: 1.
Proteins according to the invention are preferably distinguished in that on the amino acid level they have a homology of at least 95%, compared with the amino acid sequence shown in SEQ ID No: 1 and 2.
The gene PLK (polo-like kinase) described here and coding for the protein according to the invention was isolated from a cDNA bank, based on human
Holtrich Uwe
R{umlaut over (u)}bsamen-Waigmann Helga
Strebhardt Klaus
Bayer Aktiengesellschaft
Longton Enrique D.
Norris McLaughlin & Marcus P.A.
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