The cells of eukaryotes, including humans and other mammals, replicate themselves by carrying out an ordered sequence of events, which are cyclically repeated in each successive cell division. In somatic (non germ-line) cells, a typical cycle has four characterized phases: G1, an interval following the completion of mitosis, also termed first gap phase; S, a period during which the cell undergoes DNA synthesis; G2 or second gap phase following completion of DNA synthesis and preceding mitosis; and M, mitosis, where separation of complete sets of replicated DNA occurs. The end result of this process is the generation of two daughter cells that are equivalent both in genetic makeup and in size to the original parent cell. A complex series of biochemical interactions act to control the cell cycle through a series of checkpoints or gating reactions which function to ensure that the requisite precursor phases are completed before the ensuing phase begins. In particular, the checkpoints ensure accurate reproduction and dispersion of the cell's genetic material. In a metazoan organism with differentiated tissues, such as a human being, cells of different tissues replicate at vastly different rates at different life stages. Early embryonic cells replicate rapidly and synchronously, whereas at later stages of development and during adulthood, some cells, such as muscle and nerve cells stop replicating while others, such as epithelial cells, continue to divide throughout the organism's life. Failure of cells to precisely control their replicative state therefore leads to a variety of diseases of pathological proliferation, including cancers and atherosclerosis.
Progress over the past several years has greatly advanced the general understanding of the biochemical reactions which regulate the cell cycle. A general paradigm for cell cycle regulation has emerged in which complexes composed of cyclins and cyclin-dependent kinases (CDKs) regulate progression through stages of the cell cycle. Several mechanisms exist to keep the activity of the cyclin/CDK complexes turned off until the appropriate stage of the cell cycle. Known mechanisms include reversible phosphorylation, binding to small molecular weight inhibitors, transcription control, intracellular location and protein degradation. In yeast, there are multiple cyclins but only a single CDK. The CDK of fission yeast is encoded by the cdc 2 gene, that of budding yeast by the CDC28 gene. In higher eukaryotes, including humans, there are multiple CDKs as well as multiple cyclins. Despite the greater complexity of the higher eukaryotes, the overall scheme for cell cycle progression involving cyclins and CDKs is conserved. Deregulation of components of these regulatory pathways has been implicated in human cancer. For a recent general review, see Hunter, T. et al. (1994) Cell 79:573-582.
Kinase is the term applied to a class of enzyme-catalyzed reactions, many of which are well-known and described in standard collegiate biochemistry texts. In general, the reaction can be abbreviated as ##STR1## where R--OH denotes a protein or peptide having a free hydroxyl side chain (from a serine, tyrosine or threonine residue), ATP and GTP are adenosine triphosphate and guanosine triphosphate, respectively, R--OP is the protein or peptide having the free hydroxyl replaced by a phosphate ester, ADP and GDP are adenosine and guanosine diphosphate, respectively. The reaction is termed a phosphorylation reaction, the product R--OP is termed the phosphorylated form of R. The phosphate ester may be subject to enzyme-catalyzed hydrolysis, the enzymes catalyzing such hydrolysis being termed phosphatases. The phosphatase catalyzed reaction can be diagrammed as ##STR2## where Pi denotes an inorganic phosphate ion. A critical feature of the phosphatase and kinases discussed herein is their extreme specificity. A given kinase is often specific only for generating a phosphate ester on a single amino acid residue on a single protein. The same can be true of a phosphatase.
Of particular importance herein is the regulation of entry into mitosis: passage from G2 to M. The Cdc2 kinase must be enzymatically active to catalyze the phosphorylation of several cellular proteins, some structural and others regulatory. The combined functions of these phosphorylated proteins lead to exit from G2 and entry into mitosis.
Early in the cell cycle, Cdc2 exists in an underphosphorylated, monomeric form that is inactive as a protein kinase. As cells progress into S-phase, cyclin B accumulates and assembles with Cdc2 to form a complex, also inactive, but subject to phosphorylation itself. Three sites on the Cdc2 moiety of the Cdc2/cyclin B complex are of functional significance when phosphorylated: tyrosine 15, threonine 14 and threonine 161. Phosphorylation of the latter (Thr 161) activates the Cdc2/cyclin B complex. The enzyme that phosphorylates at Thr 161 is termed CAK/M015. However, phosphorylation at Thr 14 and Tyr 15 overrides the activation effect of phosphorylation at Thr 161, so that the complex phosphorylated at all three sites remains enzymatically inactive. In late G2, a specific phosphatase (Cdc 25) removes phosphates at Thr 14 and Tyr 15 (but not Thr 161), generating an active Cdc2/cyclin B complex which then initiates the phosphorylation of other proteins that results in entry into mitosis.
The human Cdc2/cyclin B complex is phosphorylated at both Tyr 15 and Thr 14. The human Wee 1 kinase was found to be unable to phosphorylate Cdc2/cyclin B at Thr 14. Another enzyme was therefore responsible for the phosphorylation at Thr 14 (Parker, L. L. et al. (1992) Science 257:1955-1957). Subsequently, that enzyme was identified as a dual-specificity kinase, able to phosphorylate at both Thr 14 and Tyr 15 on Cdc2/cyclin B. The enzyme was partially purified (Atherton-Fessler, S. et al. (1994) Mol. Biol. Cell 5:989-1001).
The present invention relates to the cloning of the human DNA encoding the dual specificity kinase of Tyr 15, Thr 14 on Cdc2/cyclin B, now termed Myt1Hu. A recent paper describing a Myt1 cDNA of Xenopus has been published by Mueller, P. R., et al. (1995) Science 270:86-90.
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