Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-26
2003-12-02
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S900000, C435S007100, C435S007400, C435S007700, C435S007710, C435S007720, C435S007900, C435S021000, C435S188000, C436S537000, C436S544000, C436S546000, C536S025320
Reexamination Certificate
active
06656696
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to monitoring of phosphorylation or dephosphorylation of a protein.
BACKGROUND OF THE INVENTION
The post-translational modification of proteins has been known for over 40 years and since then has become a ubiquitous feature of protein structure. The addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells. Such additions include, but are not limited to, protein phosphorylation and dephosphorylation.
Phosphorylation is a well-studied example of a post-translational modification of proteins. There are many cases in which polypeptides form higher order tertiary structures with like polypeptides (homo-oligomers) or with unalike polypeptides (hetero-oligomers). In the simplest scenario, two identical polypeptides associate to form an active homodimer. An example of this type of association is the natural association of myosin II molecules in the assembly of myosin into filaments.
The dimerization of myosin II monomers is the initial step in seeding myosin filaments. The initial dimerization is regulated by phosphorylation, the effect of which is to induce a conformational change in myosin II secondary structure resulting in the folded 10S monomer subunit extending to a 6S molecule. This active molecule is able to dimerize and subsequently to form filaments. The involvement of phosphorylation of myosin II in this priming event is somewhat controversial. Although in higher eukaryotes the conformational change is dependant on phosphorylation, in Ancanthoamoeba, a lower eukaryote, the post-translational addition of phosphate is not required to effect the initial dimerization step. It is of note that the dimerization domains in myosin II of higher eukaryotes contain the sites for phosphorylation and it is probable that phosphorylation in this region is responsible for enabling myosin II to dimerize and subsequently form filaments. In Dictyostelium this situation is reversed in that the phosphorylation sites are outside the dimerization domain and phosphorylation at these sites is required to effect the disassembly of myosin filaments. In contrast to both these examples, Acanthoamoeba myosin II is phosphorylated in the dimerization domain but this modification is not necessary to enable myosin II monomers to dimerize in this species.
By far the most frequent example of post-translational modification is the addition of phosphate to polypeptides by specific enzymes known as protein kinases. These enzymes have been identified as important regulators of the state of phosphorylation of target proteins and have been implicated as major players in regulating cellular physiology. For example, the cell-division-cycle of the eukaryotic cell is primarily regulated by the state of phosphorylation of specific proteins, the functional state of which is determined by whether or not the protein is phosphorylated. This is determined by the relative activity of protein kinases which add phosphate and protein phosphatases which remove the phosphate moiety from these proteins. Clearly dysfunction of either the kinases or phosphatases may lead to a diseased state. This is best exemplified by the uncontrolled cellular division shown by tumor cells. The regulatory pathway is composed of a large number of genes that interact in vivo to regulate the phosphorylation cascade that ultimately determines if a cell is to divide or arrest cell division.
Currently there are several approaches to analysing the state of modification of target proteins in vivo:
1. In vivo incorporation of labeled (for example, radiolabeled) phosphate, which is added to target proteins. According to one common procedure, intracellular ATP pools are labeled with
32
PO
4
, which is subsequently incorporated into protein. Analysis of modified proteins is typically performed by electrophoresis and autoradiography, with specificity enhanced by immunoprecipitation of proteins of interest prior to electrophoresis.
2. Back-labeling. The incorporation of a labeled phosphate (e.g.,
32
P) into a protein in vitro to estimate the state of modification in vivo.
3. The use of cell-membrane-permeable protein kinase inhibitors (e.g., Wortmannin, staurosporine) to block phosphorylation of target proteins.
4. Western blotting, of either 1- or 2-dimensional gels bearing test protein samples, in which phosphorylation is detected using antibodies specific for phosphorylated forms of target proteins.
5. The exploitation of eukaryotic microbial systems to identify mutations in protein kinases and/or protein phosphatases.
These strategies have certain limitations. Monitoring states of phosphorylation by pulse or steady-state labeling is merely a descriptive strategy to show which proteins are phosphorylated when samples are separated by gel electrophoresis and visualized by autoradiography. This is unsatisfactory, due to the inability to identify many of the proteins that are phosphorylated. A degree of specificity is afforded to this technique if it is combined with immunoprecipitation; however, this is of course limited by the availability of antibodies to target proteins. Moreover, only highly-expressed proteins are readily detectable using this technique, which may fail to identify many low-abundance proteins, which are potentially important regulators of cellular functions.
The use of kinase inhibitors to block activity is also problematic. For example, very few kinase inhibitors have adequate specificity to allow for the unequivocal correlation of a given kinase with a specific kinase reaction. Indeed, many inhibitors have a broad inhibitory range. For example, staurosporine is a potent inhibitor of phospholipid/Ca
+2
dependant kinases. Wortmannin is some what more specific, being limited to the phosphatidylinositol-3 kinase family. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible.
Monoclonal antibodies directed against phosphorylated epitopes, except in specific cases, exhibit a limitation of specificity comparable to that observed when in vivo labeling is undertaken. Immunological methods can only detect phosphorylated proteins globally (e.g., an anti-phosphotyrosine antibody will detect all tyrosine-phosphorylated proteins) and can only describe a steady state, rather provide a real-time assessment of protein:protein interactions. Such assays also require considerable manpower for processing.
Finally, yeast (
Saccharomyces cervisiae
and
Schizosaccharomyces pombe
) has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein kinases have been elucidated. However, these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
Recent research into the sites of protein phosphorylation has revealed a number of sequence specific motifs which, when phosphorylated or dephosphorylated, promote interaction with selected target proteins to either induce or inhibit activity of either the phosphorylated polypeptide or the target polypeptide.
For example, and not by way of limitation, many proteins involved in intracellular signal transduction have been shown to contain a domain comprising a sequence of approximately 100 amino acids; this sequence is termed the Src homology two (SH2) domain. SH2 domains bind target polypeptides that contain phosphorylated tyrosine. This binding is dependent on the primary amino acid sequence around the phosphotyrosine in the target protein and several peptide sequences which, when phosphorylated, bind to an SH2 domain have been identified (see e.g., Songyang et al., 1993
Cell,
72: 767-778). Non-limiting examples of such sequences include FLPVPEYINQS
Colyer John
Craig Roger K.
Cyclacel
Hines Ja'Na
Palmer & Dodge LLP
Smith Lynette R. F.
Williams Kathleen M.
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