Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-10-24
2004-10-12
Prouty, Rebecca (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S194000, C530S300000, C530S324000, C530S326000, C530S327000
Reexamination Certificate
active
06803203
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to compositions and methods useful for detecting the presence and quantifying the amount of double-stranded DNA-activated protein kinase (DNA-PK) in a biological sample and a method of monitoring DNA-PK activity or DNA-PK activity state in living cells.
2. Background of the Related Art
Eukaryotic cells contain many different protein kinases, each having specific functions and properties. Protein kinases are important signal transduction enzymes that regulate many aspects of cell metabolism and cell growth in eukaryotic cells. Protein kinases alter the properties of other enzymes or structural proteins by transferring a phosphate from a donor molecule, often ATP, to one or more acceptor amino acids of a substrate protein. Although the basic amino acids lysine, arginine and histidine can be phosphorylated by certain protein kinases which are primarily associated with bacterial organisms, the most common and more widely recognized phosphate acceptor amino acids in protein substrates in eukaryotic organisms are serine, threonine and/or tyrosine residues. The most common and more well-studied protein kinases are either protein-serine/threonine kinases, which are capable of transferring phosphate groups from donor molecules to serine and threonine acceptor amino acid residues in substrate proteins, or protein-tyrosine kinases, which are capable of transferring phosphate groups from donor molecules to tyrosine acceptor amino acid residues in substrate proteins.
Protein kinases have been found to modulate the activities of proteins in cells by phosphorylating their specific protein substrates. These specific phosphorylation reactions constitute a major mechanism for regulating biochemical pathways in multicellular eukaryotic organisms. Nuclear events, metabolic processes and signal transduction at the cytoplasmic membrane are coordinated through the phosphorylation and dephosphorylation of proteins that perform and, control these processes. Transcription and DNA replication are also regulated by phosphorylation. Most replication and transcription factors are phosphorylated at several sites and often by several different protein kinases. Kinases have been described that are activated by a variety of agents including cyclic nucleotides, phorbol esters, phospholipids, calcium ions, heme groups, double-stranded RNA (dsRNA); and double-stranded DNA (dsDNA). A growing number of nuclear regulatory proteins are known to be phosphorylated, but the kinases which phosphorylate these proteins frequently remain unidentified. Furthermore, many of the kinases which have been identified are not well characterized.
The characterization of many protein kinases has been facilitated by the development of in vitro protein kinase assays that specifically and quantitatively detect a particular kinase in a biological sample. Early work in this area was performed by Glass, et al. and is reported in
Anal. Biochem
., 87:566 (1978). Glass, et al. developed a procedure for isolating phosphorylated peptides and proteins on ion exchange papers under acidic conditions that is applicable to the study of protein-serine/threonine and protein-tyrosine kinases. The method is not applicable to the study of protein kinases that phosphorylate the basic amino acids because the bond between the phosphate group and the basic amino acid is hydrolyzed in the acidic conditions of the method. The paper-binding method was applied to protein kinase, assays that utilized synthetic peptides with amino acid sequences corresponding to the primary amino acid sequence at the phosphorylation sites of native proteins. Synthetic peptide substrates and methods for their rapid separation from kinase assay reaction mixtures have been useful in the detection, quantitation and characterization of numerous protein kinases.
Studies using substrate proteins and synthetic peptide substrates enabled the demonstration that a given protein kinase, whether of the protein-serine/threonine or the protein-tyrosine kinase class, did not phosphorylate all acceptor amino acid residues in a protein or peptide substrate, but was capable of specific selection of the serine, threonine or tyrosine that was to be phosphorylated in its various protein and peptide substrates. These studies lead to the determination that each protein kinase exhibited a specific phosphorylation site consensus sequence motif requirement for selection of the acceptor amino acid (phosphorylation site) in protein substrates or synthetic peptide substrates. Phosphorylation site consensus sequence motifs are comprised of a phosphorylation site acceptor amino acid (serine, threonine or tyrosine) embedded in a sequence or arrangement of amino acids that is specifically recognized by the protein kinase such that the kinase transfers the phosphate to that acceptor amino acid rather than to serines, threonines or tyrosines that are found elsewhere in the substrate but which are not so embedded. Pearson, et al. in
Methods Enzymol
., 220:62 (1991) tabulated protein kinase phosphorylation site consensus sequence motifs for over 240 protein-serine/threonine and protein-tyrosine kinases. That tabulation was derived from studies of protein kinases utilizing assays containing protein substrates or assays containing synthetic peptide substrates that were synthesized as analogs of natural phosphorylation site sequences.
Synthetic peptide analogs of phosphorylation site consensus sequence motifs are useful for detecting, quantifying and characterizing protein kinases. Synthetic peptide substrates have played an important role in the study of protein kinase (PK) substrate specificity as well as in the measurement of protein kinase activities in cell extracts. The major goals of designing synthetic peptide substrates for protein kinases are to construct peptides that have excellent kinetic properties and a high degree of specificity (Kemp, et al, 1991
, Methods Enzymol
., 200:121; Pearson, et al., 1991
, Methods Enzymol
., 200:62).
While synthetic peptides with amino acid sequences corresponding to the primary amino acid sequence at the phosphorylated sties of proteins have been found to serve as specific substrates for certain protein kinases, the degree of specificity of synthetic peptide substrates varies widely. A number of synthetic peptides act as substrates for multiple protein kinases. For example, phosphorylate kinase, protein kinase C, and the multi-functional calmodulin-dependent protein kinase all phosphorylate Ser
7
in a glycogen synthase peptide.
Synthetic peptide substrates for protein kinases are particularly useful if they are specifically phosphorylated only by the protein kinase of interest. A specific synthetic peptide substrate comprises a kinase-specific phosphorylation site consensus sequence motif and sufficient additional amino acids (amino acid spacer sequences) so as to create a peptide providing excellent kinetic properties in an assay. Preferably, a specific synthetic peptide substrate provides a kinase-specific phosphorylation site consensus sequence motif and sufficient amino acid spacer sequences so as to provide excellent kinetic properties in an assay and which amino acid spacer sequences do not provide another phosphorylation site consensus sequence motif. Additionally preferable is a specific synthetic peptide substrate providing a kinase-specific phosphorylation site consensus sequence motif, sufficient amino acid spacer sequences to provide excellent kinetic properties in an assay that do not provide another phosphorylation site consensus sequence motif and which amino acid spacer sequences do not contain another phosphate acceptor amino acid. To provide for economic synthetic procedures, it is especially preferable that a specific synthetic peptide substrate be as short as possible while providing the features described above.
Recently, a kinase that undergoes activation by linear double stranded (ds) DNA was discovered. This kinase is now known as the double-stranded DNA-activated (
Anderson Carl W.
Connelly Margery A.
Bogosian Margaret C.
Brookhaven Science Associates
Prouty Rebecca
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