Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-10-06
2002-11-05
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S350000, C530S324000, C514S002600, C435S320100, C435S252300, C435S194000, C435S006120, C435S015000, C435S091100, C536S023100
Reexamination Certificate
active
06476193
ABSTRACT:
FIELD OF THE INVENTION
The present invention disclosed herein relates to complexes of the Nlk1 protein with other proteins, in particular, complexes of the Nlk1 protein with the following proteins: TrkA, protein phosphatase-1&agr;, 14-3-3&egr;, &agr;-tropomyosin, vimentin, p0071, Ini-1, IP-1 (an intermediate filament associated protein), IP-2 (a tropomyosin homolog protein), IP-3 (a ubiquitin hydroxylase homolog protein), IP-4 (a collagen homolog protein) and IP-5 (a tropomyosin homolog protein). In addition, the present invention relates to the production of antibodies to the aforementioned Nlk1 protein complexes, and their use in, inter alia, screening, diagnosis, prognosis and therapy. The present invention further relates to the IP-1, IP-3, IP-4, and IP-5 genes and proteins, as well as derivatives, fragments, analogs and homologs, thereof.
BACKGROUND OF THE INVENTION
It is a well-established tenet in molecular biology that loss of control of cell proliferation may lead to severe diseases and disorders (e.g., neoplasia). Hence, the elucidation of the intricacies of the cell-cycle, and its deregulation during oncogenesis, will provide novel opportunities in the prophylactic, diagnostic and therapeutic management of cancer and other proliferation-related diseases. A better understanding of the cell-cycle could be achieved by the elucidation of the interactions of the various protein complexes, whose levels and biological activities are regulated through the cell-cycle. The identification and classification of these protein complexes will be useful in the development of treatment modalities and assays for various pathological processes including, but not limited to, hyperproliferative disorders (e.g., tumorigenesis and tumor progression), as well as other related genetic disorders.
It should be noted that the citation of a reference herein should not be construed as an admission that such is prior art to the present invention.
(1) The Nlk1 Protein
The Nlk1 protein (GenBank Acc. No. U11050) is a human homolog of the filamentous fungus
Aspergillus nidulans
mitotic regulator, NIMA kinase. The Nlk1 protein is a 48 Kdal serine/threonine-specific kinase which plays a key role in cell-cycle events leading to the onset of mitosis. The protein levels and activity of Nlk1 protein are regulated through the cell-cycle (see e.g., Schultz, et al., 1994
. Cell Growth Diff
. 5:625-632; Fry, et al., 1995
. J. Biol Chem
. 270:12899-12905). Analysis of the biochemical properties and in vitro substrate-specificity of Nlk1 protein have revealed striking similarities, but also some differences, between human Nlk1 protein and the fungal mitotic regulator, NIMA kinase. See e.g., Fry, et al., 1995
. J. Biol. Chem
. 270:12899-12905. Nlk1 protein is expressed during specific stages of the cell-cycle; with low levels of expression during mitosis (M) phase and in early gap phase (G1), and expression peaking during the DNA-synthesis (S) and late gap phase (G2) to reach a plateau in late G2 and M-phase. See e.g., Id. Accordingly, Nlk1 protein may function during an earlier phase of the cell-cycle than NIMA kinase, which displays maximum protein and activity levels during mitosis. See e.g., Osmani, et al., 1991
. EMBO J.
10:2669-267; Pu, et al., 1995
. J. Biol Chem
. 270:18110-18116. The Nlk1 protein is associated with the centrosome throughout the cell-cycle, including all stages of mitosis, independent of microtubules. See e.g., Fry, et al., 1998
. EMBO J
. 17:470-481. Thus, one biological function of Nlk1 protein relates to the centrosome cycle.
In certain aspects, the Nlk1 protein appears to function in a similar manner to NIMA kinase. See generally, Pu, et al., 1995
. J. Biol. Chem
. 270:18110-18116. The activity of NIMA kinase is essential for the progression of cells into mitosis, and the full activation of the NIMA kinase depends on the cyclin-dependent kinase, CDC2. Both NIMA and CDC2 kinases have been demonstrated to be required for the progression from G2 to mitosis in Aspergillus, and following this cell-cycle progression, both kinases are rapidly degraded. See e.g., Pu & Osmani, 1995
. EMBO J
. 14:995-1003. Recent experimental evidence has demonstrated that the Aspergillus NIMA serine/threonine kinase is not only required for mitosis, in cooperation with CDC2, but is also implicated in chromatin condensation. See e.g., Lu & Hunter, 1995
. Cell
81:413-424; Pu & Osmani, 1995
. EMBO J
. 14:993-1003. Additionally, the Nlk1 protein may also be involved in other events of meiosis including, but not limited to, chromosomal condensation. See e.g., Rhee & Wolgemuth, 1997
. Development
124:2167-2177.
As previously discussed, CDC2 is responsible for the phosphorylation of the Aspergillus NIMA kinase (see Fry & Nigg, 1995
. Curr. Biol
. 5:1122-1125), interestingly, however, mammalian Nlk1 protein lacks the putative CDC2 phosphorylation sites. While Nlk1 protein has been shown to be active as a serine/threonine kinase, the precise sites which are phosphorylated by Nlk1 protein have not yet been mapped. Recently, a synthetic peptide possessing the amino acid sequence, IRRLSTRR, was found to be phosphorylated exclusively on serine residues, thus tending to suggest that basic amino acid residues may contribute to substrate recognition by Nlk1 protein. See e.g., Fry, et al., 1995
. J. Biol. Chem
. 270:12899-12905.
The Nlk1 protein possesses marked sequence homology with NIMA kinase, with the two kinases sharing a 48% sequence identity over their catalytic domains located at the amino-terminus. See e.g., Schultz, et al., 1994
. Cell Growth Diff
. 5:625-635. Another conserved feature of the NIMA-related kinase family is the position of an insertion (maximal 25 amino acids) between two kinase the subdomains, VIA and VIB. Interestingly, the carboxyl-terminal extensions display virtually no primary sequence conservation among the different members of the NIMA-related kinase family. However, the beginning of the highly basic carboxyl-terminal, non-catalytic extension contains a conserved secondary structural motif (i.e., a coiled-coil) in both NIMA kinase and Nlk1 protein, which functions in the degradation of NIMA kinase during the M-phase. Coiled-coil motifs are often found to be involved in protein-protein interaction, and such secondary structural motifs may have been conserved as a direct result of their putative biological function(s). See e.g., Fry & Nigg, 1997
. Meth. Enzym
. 283:270-282. The carboxyl-terminal non-catalytic domain within human Nlk1 protein are markedly shortened, and it is interesting to note that several putative CDC2 phosphorylation sites found within the carboxyl-terminal domain of NIMA, are not found to be present in the much shorter extension of Nlk1 protein.
The Aspergillus NIMA kinase interacts with CDC2-kinase (see e.g., Osmani, et al, 1994
. J. Cell Sci
. 107:1519-1528) and with the human Pin1 protein, a peptidyl-prolyl-isomerase (see e.g., Lu, et al, 1996
. Nature
380:544-547). Heretofore the present invention, no interacting proteins have been described for the human NIMA kinase homologue Nlk1 protein. The Nlk1 protein differs from NIMA kinase in that: (i) Nlk1 protein activity peaks in the cell-cycle's S/G2 phase, and not in mitosis; (ii) Nlk1 protein lacks the putative CDC2 phosphorylation sites found in NIMA kinase and (iii) Nlk1 protein lacks the carboxyl-terminal PEST sequences found in NIMA kinase. Hence, the exact biological function(s) of the Nlk1 protein have yet to be elucidated, although along with the other members of the Nek family, it almost certainly functions in some form of cell-cycle mediation and control.
(2) Nlk1 protein-Interacting Proteins
The Nlk1 protein, a serine/threonine-specific kinase, plays an important role in cell-cycle events which lead to the onset of mitosis. Furthermore, Nlk1 protein is also involved in chromosome condensation and thus, in events of meiosis. Despite these aforementioned roles, heretofore the present invention, there has been no quantitative data regarding the interaction of the Nlk1 protein with other cellular proteins
Nandabalan Krishnan
Schulz Vincent P.
Yang Meija
Biswas Naomi S.
Carlson Karen Cochrane
CuraGen Corporation
Elrifi Ivor R.
Kam Chih-Min
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