Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-06-21
2001-05-01
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C530S350000
Reexamination Certificate
active
06225101
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the cell cycle and mitosis. More specifically, the invention relates to a threonine and tyrosine protein kinase, Myt1, that is a Cdc2 inhibitory kinase regulated during the cell cycle in such a way that it plays a role in mitotic control.
BACKGROUND OF THE INVENTION
The replication cycle of a typical eukaryotic somatic cell consists of four phases: G
1
, S (DNA synthesis), G
2
, and M (mitosis). The result of this process is the generation of two daughter cells that are equivalent both in genetic makeup and in size to the original parental cell. Feedback controls operating at checkpoints ensure the faithful replication and segregation of the genetic material. In eukaryotic organisms, a general paradigm has emerged in which a family of proteins, called cyclins, and cyclin-dependent protein kinases (Cdks) regulate cell cycle progression. These mechanisms are at the level of reversible phosphorylation, binding to low-molecular-weight inhibitors, transcription, intracellular compartmentalization, and protein degradation.
The transition from G
2
to M phase requires the activity of M-phase-promoting factor (MPF), which is composed of Cdc2, an evolutionarily conserved serine/threonine-specific protein kinase, and B-type cyclins. The activity of Cdc2 is regulated not only by is association with B-type cyclins but also by reversible phosphorylation. Proper regulation of MPF ensures that mitosis occurs only after earlier phases of the cell cycle have been completed successfully. This strict control of MPF is largely post-translational, involving the phosphorylation of Cdc2 at three key residues. After Cdc2 associates with cyclin, the cyclin-dependent kinase (CDK)-activating kinase (CAK) phosphorylates Cdc2 on Thr
161
. This phosphorylation would generate active MPF, but two additional phosphorylations on Thr
14
and Tyr
15
of Cdc2 suppress MPF activity during interphase. At the G
2
-M transition, the Cdc25 protein dephosphorylates Thr
14
and Tyr
15
, thereby allowing MPF to phosphorylate its mitotic substrates.
Phosphorylation on Thr
14
and Tyr
15
maintains Cdc2 in an inactive state throughout the S and G
2
phases of the cell cycle, and Thr
161
phosphorylation is required for the kinase activity of the complex. Dephosphorylation of both Thr
14
and Tyr
15
by the Cdc25 phosphatase in late G
2
activates Cdc2 and is an obligate step for the onset of mitosis. Exit from mitosis requires the proteolytic degradation of the B-type cyclins, which is mediated by ubiquitination.
Various genetic and biochemical studies have indicated that Wee1 is the kinase that phosphorylates Cdc2 on Tyr
15
. Wee1 was originally identified in the fission yeast
Schizosaccharomyces pombe
as a critical negative regulator of mitosis. Subsequently, a second
S. pombe
homolog (Mik1) and Wee1 homologs from at least six other organisms have been found. In human and Xenopus, Wee1 is a soluble enzyme that phosphorylates Cdc2 on Tyr
15
, but not on Thr
14
.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both Thr
14
and Tyr
15
. Although Wee1 had been identified as the kinase that phosphorylates Tyr
15
in various organisms, the Thr
14
-specific kinase had not been identified. A complementary DNA (cDNA) cloned from Xenopus, encodes Myt1 (“membrane-associated, tyrosine-and threonine-specific Cdc2 inhibitory kinase”). Myt1 is a membrane-associated protein that contains a putative transmembrane segment.
In a first embodiment, the invention provides a substantially purified Myt1 polypeptide exemplified by the amino acid sequence of SEQ ID NO:2 and polynucleotides encoding SEQ ID NO:2. Also included are vectors and host cells containing the polynucleotide encoding Myt1 of the invention.
In another embodiment, the invention provides a method of measuring the activity of Myt1 in a sample. The method includes incubating a test sample with a substrate for Myt1 and labeled phosphate under conditions sufficient to allow phosphorylation of the substrate; and determining the rate of incorporation of labeled phosphate into the substrate, wherein the rate of incorporation is a measure of Myt1 activity. The substrate is preferably Cdc2.
In yet another embodiment, the invention provides a method for measuring the synthesis of Myt1 in a test sample. The method includes a) obtaining a biological sample; b) contacting the sample with an antibody that specifically binds an Myt1 polypeptide of the invention; and c) detecting the antibody bound to Myt1 polypeptide, wherein the level of Myt1 synthesis is determined by the amount of bound antibody. Preferably the antibody is an Myt1-specific monoclonal antibody.
In another embodiment, the invention provides a method for measuring the level of expression of Myt1 in a test sample. The method includes a) isolating total or polyadenylated RNA from the test sample; b) incubating the RNA with a polynucleotide probe specific for an Myt1 polynucleotide of the invention; and c) determining the amount of the probe hybridized to the RNA, wherein the level of expression of Myt1 is directly related to the amount of Myt1 probe hybridized to the RNA.
In another embodiment, the invention provides a method for identifying a reagent that modulates Myt1 activity. The method includes a) obtaining a test sample containing Myt1; b) incubating the test sample with a substrate for the Myt1 polypeptide of the invention, the reagent, and labeled phosphate under conditions sufficient to allow phosphorylation of the substrate in the absence of the reagent; c) detecting phosphorylation of the substrate; and d) comparing the effect of the reagent on Myt1 activity relative to a control not containing the reagent, wherein any variation compared to control is indicative of a reagent which modulates Myt1 substrate phosphorylation. Preferably, the substrate is Cdc2. The modulation measured may be either inhibition of Myt1 activity or stimulation of Myt1 activity.
In another embodiment, the invention provides a method for identifying a reagent that modulates Myt1 synthesis. The method includes a) providing a sample capable of Myt1 synthesis; b) incubating the sample with a reagent under conditions that allow synthesis of Myt1 in the absence of the reagent; c) detecting an Myt1 polypeptide of the invention; and d) comparing the effect of the reagent on Myt1 synthesis relative to a control not containing the reagent, wherein any variation compared to control is indicative of a reagent which modulates Myt1 synthesis.
In another embodiment, the invention provides a method for identifying a reagent that modulates Myt1 expression. The method includes a) providing a sample capable of expressing Myt1; b) incubating the sample with a reagent under conditions where Myt1 is expressed in the absence of the reagent; c) isolating total or polyadenylated RNA from the sample; d) incubating the RNA with a polynucleotide probe specific for a Myt1 nucleic acid of the invention; and e) comparing the effect of the reagent on Myt1 RNA expression relative to a control, wherein any variation compared to control is indicative of a reagent which modulates Myt1 expression.
In another embodiment, the invention provides a method for treating an Myt1-mediated disorder in a patient including administering to the patient a therapeutically effective amount of a reagent that modulates Myt1 activity. Also included is a method of treating an Myt1-associated disorder in a patient, including administering to the patient a therapeutically effective amount of an Myt1 polypeptide or Myt1 nucleic acid.
In a further embodiment, the invention includes a kit useful for the detection of Myt1 polypeptide or polynucleotide, the kit including a carrier means being compartmentalized to receive in close confinement therein one or more containers comprising a first container containing an antibody which binds to Myt1 polypeptide or nucleic acid probes that bind Myt1 polynucleotide, respectively, and a second container c
Coleman Thomas R.
Dunphy William G.
Kumagai Akiko
Mueller Paul R.
California Institute of Technology
Fish & Richardson P.C.
McKelvey Terry
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