Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1999-09-08
2003-01-07
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S320100, C435S252300, C435S325000, C536S023200
Reexamination Certificate
active
06503742
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Cell-cycle Regulatory Proteins
Cell-cycle events are thought to be regulated by a series of interdependent biochemical steps. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. In all eukaryotic cells examined to date, the cell cycle appears to be regulated by the sequential activation of a series of the CDK's or Cyclin Dependent Kinases (reviewed in Morgan, (1995)
Nature
374:131-134; King et al., (1994)
Cell
79:563-571; Norbury and Nurse, (1992)
Annu. Rev. Biochem.
61:441-470). Yeast cells contain a single CDK known as cdc2 in
S. pombe
(Beach et al., (1982)
Nature
300:706-709; Booher and Beach, (1986)
Gene
31:129-134; Hindley and Phear, (1984)
Gene
21:129-134; Nurse and Bissett, (1981)
Nature
292:558-560; Simanis and Nurse, (1986)
Cell
45:261-268; and for review see Forsburg and Nurse, (1991b)
Annu. Rev. Cell Riol.
7:227-256) and cdc28 in
S. cerevisae.
The similarities between the progression of proliferation in mammalian cells and yeast have suggested similar roles for cdc protein kinases across species. In support of this hypothesis, a human cdc2 gene has been found to be able to substitute for the activity of an
S. pombe
cdc2 gene in both its G1/S and G2/M roles (Lee et al., (1987)
Nature
327:31). Likewise, the fact that the cdc2 homolog of
S. cerevisae
(cdc28) can be replaced by the human cdc2 also emphasizes the extent to which the basic cell-cycle machinery has been conserved in evolution.
The activation of cdc2 kinase activity occurs during the M phase and is controlled at multiple levels involving, among other events, the association with various cyclin subunits and the phosphorylation on threonine 167 by cdc2 activating kinase (CAK) (Booher and Beach, (1987)
EMBO J.
6:3441-3447; Booher et al., (1989)
Cell
58:485-497; Bueno et al., (1991)
Cell
66:149-159; Bueno and Russell, (1993)
Mol. Cell Biol.
13:2286-2297; Connolly and Beach, (1994)
Mol. Cell Biol.
14:768-776; Fesquet et al., (1993)
EMBO J.
12:3111-3121; Forsburg and Nurse, (1991a)
Nature
351:245-247; Gould et al., (1991)
EMBO J.
3297-3309; Hagan et al., (1988)
J. Cell Sci.
91:587-595; Solomon et al., (1992)
Mol. Biol. Cell
3:13-27; Solomon et al., (1993)
EMBO J.
12:3133-3142). Another well-characterized mechanism of regulating the activity of cdc2 involves its inhibition by phosphorylation of a tyrosine and threonine residues (Tyr-15 and Thr-14) within its ATP binding site (Gould and Nurse, (1989) supra). The inhibitory phosphorylation of cdc2 is mediated at least impart by the weel and mik1 tyrosine kinases (Russel et al., (1987)
Cell
49:559-567; Lundgren et al., (1991)
Cell
64:1111-1122; Featherstone et al., (1991)
Nature
349:808-811; and Parker et al., (1992)
PNAS
89:2917-2921). These kinases act as mitotic inhibitors, over-expression of them causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al., (1991)
Cell
64:1111-1122).
As the cell is about to reach the end of G2, dephosphorylation of the cdc2-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the cdc2 complex as a kinase. With the exception of budding yeast and the early embryonic cell divisions of some organisms, the dephosphorylation of tyrosine 15 is a key regulatory step of cdc2 activation (Morla et al., (1989)
Cell
58:193-203; Heald et al., (1993)
Cell
74:463-474; and for reviews see King et al., (1994)
Cell
79:563-571; and Morgan (1995)
Nature
374:131-134). A stimulatory phosphatase, known as cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al., (1991)
Cell
67:189-196; Lee et al., (1992)
Mol Biol Cell
3:73-84; Millar et al., (1991)
EMBO J
10:4301-4309; and Russell et al., (1986)
Cell
45:145-153). Cdc25 has been shown to be required for entry into mitosis in a number of different organisms (King et al., 1994). Evidence indicates that both the cdc25 phosphatase and the cdc2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al., (1992)
Cell
70:139-151; Smythe et al., (1992)
Cell
68:787-797; and Solomon et al., (1990)
Cell
63:1013-1024. This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of cdc2 which is likely controlled by variation of the rate of tyrosine dephosphorylation of cdc2 and/or a decrease in the rate of its tyrosine phosphorylation.
In
S. pombe,
the level of cdc25 oscillates in a cell cycle dependent fashion (Ducommum et al., (1990)
Biochem. Biophys. Res. Comm.
167:301-309; Moreno et al., (1990)
Nature
344:549-552). Cdc25 accumulates through the cell cycle until mitosis when its level rapidly decreases. The pattern of cdc25 accumulation during the cell cycle is reminiscent of mitotic cyclins which are degraded by the ubiquitin system (Glotzer et al., (1991)
Nature
349:132-138; Seufert et al., (1995)
Nature
373:78-81).
(b) Ubiquitination Pathways
The ubiquitin-mediated proteolysis system is the major pathway for the selective, controlled degradation of intracellular proteins in eukaryotic cells. Ubiquitin modification of a variety of protein targets within the cell appears to be important in a number of basic cellular functions such as regulation of gene expression, regulation of the cell-cycle, modification of cell surface receptors, biogenesis of ribosomes, and DNA repair. One major function of the ubiquitin-mediated system is to control the half-lives of cellular proteins. The half-life of different proteins can range from a few minutes to several days, and can vary considerably depending on the cell-type, nutritional and environmental conditions, as well as the stage of the cell-cycle.
Targeted proteins undergoing selective degradation, presumably through the actions of a ubiquitin-dependent proteosome, are covalently tagged with ubiquitin through the formation of an isopeptide bond between the C-terminal glycyl residue of ubiquitin and a specific lysyl residue in the substrate protein. This process is catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), and in some instances may also require auxiliary substrate recognition proteins (E3s). Following the linkage of the first ubiquitin chain, additional molecules of ubiquitin may be attached to lysine side chains of the previously conjugated moiety to form branched multi-ubiquitin chains.
The conjugation of ubiquitin to protein substrates is a multi-step process. In an initial ATP requiring step, a thioester is formed between the C-terminus of ubiquitin and an internal cysteine residue of an E1 enzyme. Activated ubiquitin is then transferred to a specific cysteine on one of several E2 enzymes. Finally, these E2 enzymes donate ubiquitin to protein substrates. Substrates are recognized either directly by ubiquitin-conjugated enzymes or by associated substrate recognition proteins, the E3 proteins, also known as ubiquitin ligases.
Many proteins that control cell-cycle progression are short-lived. For example, regulation of oncoproteins and anti-oncoproteins clearly plays an important role in determining steady-state levels of protein expression, and alterations in protein degradation are as likely as changes in transcription and/or translation to cause either the proliferative arrest of cells, or alternatively, the transformation of cells.
SUMMARY OF THE INVENTION
The present invention relates to the discovery in eukaryotic cells of novel family of proteins whose apparent function includes a ubiquitin ligase activity. In particular, one feature of members of this family of proteins includes a catalytic domain containing a regio
Beach David
Caligiuri Maureen G.
Nefsky Bradley
Cold Spring Harbor Laboratory
Prouty Rebecca E.
Ropes & Gray
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