Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-07-17
2001-04-10
Eyler, Yvonne (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S069100, C435S325000, C435S173300, C435S252300, C536S024300, C536S023100
Reexamination Certificate
active
06214562
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a G protein-coupled receptor whose expression is regulated in hematopoietic cells and functions at the G2/M checkpoint to ensure properly controlled duplication of hematopoietic cells.
BACKGROUND OF THE INVENTION
The family of G protein-coupled receptors (GPCRs) has at least 250 members (Strader et al.
FASEB J.,
9:745-754, 1995; Strader et al.
Annu. Rev. Biochem.,
63:101-32, 1994). It has been estimated that one percent of human genes may encode GPCRs. GPCRs bind to a wide-variety of ligands ranging from photons, small biogenic amines (i.e., epinephrine and histamine), peptides (i.e., IL-8), to large glycoprotein hormones (i.e., parathyroid hormone). Upon ligand binding, GPCRs regulate intracellular signaling pathways by activating guanine nucleotide-binding proteins (G proteins). GPCRs play important roles in diverse cellular processes including cell proliferation and differentiation, leukocyte migration in response to inflammation, and cellular response to light, odorants, neurotransmitters and hormones (Strader et al., supra.).
Interestingly, GPCRs have functional homologues in human cytomegalovirus and herpesvirus, suggesting that GPCRs may have been acquired during evolution for viral pathogenesis (Strader et al.,
FASEB J.,
9:745-754, 1995; Arvanitakis et al.
Nature,
385:347-350, 1997; Murphy,
Annu. Rev. Immunol.
12:593-633, 1994).
The importance of G protein-coupled receptors is further highlighted by the recent discoveries that its family members, chemokine receptors CXCR4/Fusin and CCR5, are co-receptors for T cell-tropic and macrophage-tropic HIV virus strains respectively (Alkhatib et al.,
Science,
272:1955, 1996; Choe et al.,
Cell,
85:1135, 1996; Deng et al.,
Nature,
381:661, 1996; Doranz et al.,
Cell,
85:1149, 1996; Dragic et al.,
Nature,
381:667 (1996); Feng et al.,
Science
272:872, 1996). It is conceivable that blocking these receptors may prevent infection by the human immunodeficiency (HIV) virus.
Cell cycle checkpoints, intervals in the cell cycle in which the cell detects impairment or loss of integrity to its genome and arrests growth in order to make repairs, ensure that DNA is replicated with high fidelity (Paulovich et al.,
Cell
88:315-321, 1997; Hartwell,
Cell
71:543-546, 1992). There are three separately defined times in the eukaryotic cell cycle identified as checkpoints: G1/S transition, S-phase delay and G2/M transition (Nurse,
Cell
91:865-867, 1997). The G1/S checkpoint is activated to avoid copying mutated DNA by increasing the time available for repair. Cells also utilize a DNA damage checkpoint within S phase by slowing the rate of DNA replication. The G2/M checkpoint is activated upon detection of double-stranded DNA breaks. In addition, mitotic entry is monitored by a spindle checkpoint that inhibits anaphase progression when chromosomes are not attached to the mitotic spindle (Nicklas,
Science
275:632-637, 1997). The cell cycle checkpoint is summarized in FIG.
1
.
Recent discoveries have shed light on the molecular participants in the G2/M transition. Cdc2 and Cyclin B1 promote entry into mitosis and are part of the maturation promoting factor (MPF). Dephosphorylation of Cdc2 on Thr14 and Tyr15 by Cdc25 and phosphorylation on Thr161 concomitant with nuclear association with Cyclin B1 results in rapid entry into mitosis. Cyclin B1 degradation or export to the cytoplasm and phosphorylation of Cdc2 on the negative regulatory sites Thr14 and Tyr15 by Weel block entry into mitosis. Caffeine can relieve DNA damage-activated G2/M arrest by stimulating the dephosphorylation of Cdc2. These data strongly implicate MPF as the central regulator of the transition from G2 into mitosis.
Recent work has broadened our understanding of the signaling pathways involved in G2/M arrest upstream of MPF. Response to DNA damage is detected by the Ataxia Telangiectasia mutated (ATM) which is a human homologue of the yeast rad family of genes (Meyn, 1995). The ATM protein has been implicated in the activation of Chk1, which phosphorylates Cdc25, leading to binding and sequestering of Cdc25 by 14-3-3 (Sanchez et al.,
Science
277:1497-1501, 1997; Peng et al.,
Science
277:1501-1505, 1997; Furnari,
Science
277:1495-1497, 1997). This results in accumulation of the phosphorylated (inactive) form of Cdc2 and G2/M arrest. Cds1 has been demonstrated to function redundantly to Chk-1 by phosphorylating both Wee1 and Cdc25, inactivating both gene products (Boddy et al.,
Science
280:909-912, 1998; Fumari et al, supra.; Sanchez et al., supra.). ATM serves to activate proteins that act directly on MPF and lead to cell cycle arrest.
ATM also associates with and activates proteins that stimulate transcription of secondary molecules involved in checkpoint controls. One of these downstream activators of ATM is the tumor suppressor p53. Activation of p53 leads to the induction of multiple genes, including p21Cip and 14-3-3 (Levine,
Cell
88:323-331, 1997). The 14-3-3 gene product mediates G2M arrest by binding to Cdc25 to sequester it in the cytoplasm. The tyrosine kinase Abl physically interacts with the ATM gene product (Shafman,
Nature
387:520-523, 1997; Baskaran,
Nature
387:516-519, 1997). Activation of the Abl kinase by DNA damage is dependent on the ATM, suggesting a functional link of Abl and ATM in the DNA damage checkpoint regulation. The overall regulation of the G2M checkpoint is an intricate mechanism involving both post-transcriptional modifications and transcriptional activation to guarantee proper cell growth. Thus, the known G2/M checkpoint proteins ultimately function through regulation of Cdc2 phosphorylation and nuclear import of Cyclin B1.
While the general eukaryotic cell cycle control machinery is highly conserved among a broad range of cell types, little is known about tissue-specific cell cycle regulators. TGF-&bgr; and GATA-5 represent anti-proliferative signaling molecules that are restricted in expression. Both of these regulators restrict the cell cycle at G1. Lymphocytes provide an interesting model system to study tissue-specific cell cycle regulators since their development is marked by the unique property of entering, exiting and re-joining the cell cycle depending on their internal developmental stages as well as the surrounding environment. For example, upon interaction with antigen, the resting mature naive B cells accumulate in the lymphoid germinal centers in which they undergo vigorous proliferation and excess B cells die by being included from germinal centers.
Loss of cellular growth controls by oncogenic transformation is dependent on signals emanating from the oncogene to downstream signaling partners and frequently leads to transcriptional induction of secondary genes which contribute to malignant growth. BCR-ABL is a chimeric tyrosine kinase oncogene generated by a reciprocal chromosomal translocation t(9;22)(q34;q11) associated with the pathogenesis of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) (Kurzrock,
N. Engl. J. Med.
319: 990-998, 1988). This chimeric oncogenesis found in Ph
1
-positive stem cells. Structural and functional analysis have defined critical domains within BCR-ABL responsible for its oncogenic activity. In particular, the R552L substitution within a highly conserved motif of the Src Homology 2 (SH2) domain uncouples the SH2 domain with phosphotyrosine-containing proteins without affecting the kinase activity of BCR-ABL. Interestingly, this mutation greatly reduces the ability of BCR-ABL to stimulate anchorage-independent growth of rat fibroblasts in soft agar (Goga,
Cell
82:981-988, 1995). Although the SH2 mutant still retains the ability to transform primary bone marrow cells in vitro, it exhibits diminished malignant and leukemogenic potential in mice (Goga, supra.). Inactivation of the SH2 domain may uncouple BCR-ABL with downstream signaling molecules, which in turn may alter the expression of critical genes involved in leukemogenesis.
SUMMARY OF THE INVENTION
One embodiment of the present in
Weng Zhigang
Witte Owen N.
Basi Nirmal S.
Eyler Yvonne
Gates & Cooper LLP
The Regents of the University of California
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